Compounds and methods for modulating gfap

ABSTRACT

Provided are compounds, methods, and pharmaceutical compositions for reducing the amount or activity of GFAP RNA in a cell or subject, and in certain instances reducing the amount of GFAP in a cell or subject. Such compounds, methods, and pharmaceutical compositions are useful to ameliorate at least one symptom or hallmark of a leukodystrophy. Such symptoms and hallmarks include motor delays, cognitive delays, paroxysmal deterioration, seizures, vomiting, swallowing difficulties, ataxic gait, palatal myoclonus, autonomic dysfunction, and presence of intra-astrocytic inclusions called Rosenthal fibers. Such leukodystrophies include Alexander Disease.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0353USSEQ_ST25.txt, created on Jul. 17, 2020, which is 592 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

Provided are compounds, methods, and pharmaceutical compositions for reducing the amount or activity of GFAP RNA in a cell or subject, and in certain instances reducing the amount of glial fibrillary acidic protein (GFAP) in a cell or subject. Such compounds, methods, and pharmaceutical compositions are useful to ameliorate at least one symptom or hallmark of a leukodystrophy. Such symptoms and hallmarks include motor delays, cognitive delays, paroxysmal deterioration, seizures, vomiting, swallowing difficulties, ataxic gait, palatal myoclonus, autonomic dysfunction, and intra-astrocytic inclusions called Rosenthal fibers. Such leukodystrophies include Alexander Disease.

BACKGROUND

Alexander Disease (AxD) is a rare developmental disorder that affects ˜1/1,000,000 live births and is caused by a number of different autosomal dominant mutations in the gene encoding glial fibrillary acidic protein, GFAP. AxD is a typically fatal leukodystrophy with early onset (<age 4, Type I) or later onset (>age 4 Type II) forms (Prust et al., (2011) GFAP mutations, age at onset, and clinical subtypes in Alexander disease. Neurol 77: 1287-1294). Symptoms include motor and cognitive delays, paroxysmal deterioration, seizures, encephalopathy, macrocephaly, and intra-astrocytic inclusions called Rosenthal fibers.

There are no specific therapies for AxD, with current treatments being limited to supportive treatments for individual symptoms (e.g., antiepileptics to prevent seizures; Messing, et. al., “Strategies for treatment in Alexander Disease”, Neurotherapeutics: The Journal of the Am. Soc. For Expt. NeuroTher., 2016).

Currently there is a lack of acceptable options for treating leukodystrophies such as AxD. It is therefore an object herein to provide compounds, methods, and pharmaceutical compositions for the treatment of such diseases.

SUMMARY OF THE INVENTION

Provided herein are compounds, methods and pharmaceutical compositions for reducing the amount or activity of GFAP RNA, and in certain embodiments reducing the expression of glial fibrillary acidic protein in a cell or subject. In certain embodiments, the subject has a leukodystrophy. In certain embodiments, the subject has Alexander Disease (AxD). In certain embodiments, compounds useful for reducing the amount or activity of GFAP RNA are oligomeric compounds. In certain embodiments, compounds useful for reducing the amount or activity of GFAP RNA are modified oligonucleotides. In certain embodiments, compounds useful for decreasing expression of glial fibrillary acidic protein are oligomeric compounds. In certain embodiments, compounds useful for decreasing expression of glial fibrillary acidic protein are modified oligonucleotides.

Also provided are methods useful for ameliorating at least one symptom or hallmark of a leukodystrophy. In certain embodiments, the leukodystrophy is Alexander Disease. In certain embodiments, the symptom or hallmark includes motor delays, cognitive delays, paroxysmal deterioration, seizures, vomiting, swallowing difficulties, ataxic gait, palatal myoclonus, autonomic dysfunction, and presence of intra-astrocytic inclusions called Rosenthal fibers.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and GenBank, ENSEMBL, and NCBI reference sequence records, are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

Definitions

As used herein, “2′-deoxynucleoside” means a nucleoside comprising a 2′-H(H) deoxyfuranosyl sugar moiety. In certain embodiments, a 2′-deoxynucleoside is a 2′-β-D-deoxynucleoside and comprises a 2′-β-D-deoxyribosyl sugar moiety, which has the β-D ribosyl configuration as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

As used herein, “2′-MOE” or “2′-MOE sugar moiety” means a 2′-OCH₂CH₂OCH₃ group in place of the 2′—OH group of a furanosyl sugar moiety. A “2′-MOE sugar moiety” means a sugar moiety with a 2′-OCH₂CH₂OCH₃ group in place of the 2′—OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2′-MOE sugar moiety is in the β-D-ribosyl configuration. “MOE” means O-methoxyethyl.

As used herein, “2′-MOE nucleoside” means a nucleoside comprising a 2′-MOE sugar moiety.

As used herein, “2′-OMe” or “2′-O-methyl sugar moiety” means a 2′-OCH₃ group in place of the 2′—OH group of a furanosyl sugar moiety. A “2′-O-methyl sugar moiety” or “2′-OMe sugar moiety” means a sugar moiety with a 2′-OCH₃ group in place of the 2′—OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2′-OMe sugar moiety is in the β-D-ribosyl configuration.

As used herein, “2′-OMe nucleoside” means a nucleoside comprising a 2′-OMe sugar moiety.

As used herein, “2′-substituted nucleoside” means a nucleoside comprising a 2′-substituted sugar moiety. As used herein, “2′-substituted” in reference to a sugar moiety means a sugar moiety comprising at least one 2′-substituent group other than H or OH.

As used herein, “5-methyl cytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methyl cytosine is a modified nucleobase.

As used herein, “administering” means providing a pharmaceutical agent to a subject.

As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.

As used herein, “antisense compound” means an oligomeric compound capable of achieving at least one antisense activity.

As used herein, “ameliorate” in reference to a treatment means improvement in at least one symptom or hallmark relative to the same symptom or hallmark in the absence of the treatment. In certain embodiments, amelioration is the reduction in the severity or frequency of a symptom or the delayed onset or slowing of progression in the severity or frequency of a symptom. In certain embodiments, the symptom or hallmark is motor delays, cognitive delays, paroxysmal deterioration, seizures, vomiting, swallowing difficulties, ataxic gait, palatal myoclonus, autonomic dysfunction, and presence of intra-astrocytic inclusions called Rosenthal fibers.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.

As used herein, “bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the furanosyl sugar moiety is a ribosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.

As used herein, “cerebrospinal fluid” or “CSF” means the fluid filling the space around the brain and spinal cord. “Artificial cerebrospinal fluid” or “aCSF” means a prepared or manufactured fluid that has certain properties of cerebrospinal fluid.

As used herein, “cleavable moiety” means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.

As used herein, “complementary” in reference to an oligonucleotide means that at least 70% of the nucleobases of the oligonucleotide or one or more portions thereof and the nucleobases of another nucleic acid or one or more portions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. As used herein, “complementary nucleobases” means nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl cytosine (^(m)C) and guanine (G). Complementary oligonucleotides and/or target nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to an oligonucleotide, or a portion thereof, means that the oligonucleotide, or portion thereof, is complementary to another oligonucleotide or target nucleic acid at each nucleobase of the shorter of the two oligonucleotides, or at each nucleoside if the oligonucleotides are the same length.

As used herein, “conjugate group” means a group of atoms that is directly or indirectly attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

As used herein, “conjugate linker” means a single bond or a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

As used herein, “conjugate moiety” means a group of atoms that is attached to an oligonucleotide via a conjugate linker.

As used herein, “contiguous” in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.

As used herein, “constrained ethyl” or “cEt” or “cEt modified sugar moiety” means a 4′ to 2′ bridge in place of the 2′OH-group of a ribosyl sugar moiety, wherein the bridge has the formula of 4′-CH(CH₃)—O-2′, and wherein the methyl group of the bridge is in the S configuration. A “cEt sugar moiety” is a bicyclic sugar moiety with a 4′ to 2′ bridge in place of the 2′OH-group of a ribosyl sugar moiety, wherein the bridge has the formula 4′-CH(CH₃)—O-2′, and wherein the methyl group of the bridge is in the S configuration.

As used herein, “cEt nucleoside” means a nucleoside comprising a cEt sugar moiety.

As used herein, “chirally enriched population” means a plurality of molecules of identical molecular formula, wherein the number or percentage of molecules within the population that contain a particular stereochemical configuration at a particular chiral center is greater than the number or percentage of molecules expected to contain the same particular stereochemical configuration at the same particular chiral center within the population if the particular chiral center were stereorandom. Chirally enriched populations of molecules having multiple chiral centers within each molecule may contain one or more stereorandom chiral centers. In certain embodiments, the molecules are modified oligonucleotides. In certain embodiments, the molecules are compounds comprising modified oligonucleotides.

As used herein, “chirally controlled” in reference to an internucleoside linkage means chirality at that linkage is enriched for a particular stereochemical configuration.

As used herein, “deoxy region” means a region of 5-12 contiguous nucleotides, wherein at least 70% of the nucleosides are 2′-β-D-deoxynucleosides. In certain embodiments, each nucleoside is selected from a 2′-β-D-deoxynucleoside, a bicyclic nucleoside, and a 2′-susbstituted nucleoside. In certain embodiments, a deoxy region supports RNase H activity. In certain embodiments, a deoxy region is the gap or internal region of a gapmer.

As used herein, “gapmer” means a modified oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.” The internal region is a deoxy region. The positions of the internal region or gap refer to the order of the nucleosides of the internal region and are counted starting from the 5′-end of the internal region. Unless otherwise indicated, “gapmer” refers to a sugar motif. Unless otherwise indicated, the sugar moiety of each nucleoside of the gap is a 2′-β-D-deoxynucleoside. In certain embodiments, the gap comprises one 2′-substituted nucleoside at position 1, 2, 3, 4, or 5 of the gap, and the remainder of the nucleosides of the gap are 2′-β-D-deoxynucleosides. As used herein, the term “MOE gapmer” indicates a gapmer having a gap comprising 2′-β-D-deoxynucleosides and wings comprising 2′-MOE nucleosides. As used herein, the term “mixed wing gapmer” indicates a gapmer having wings comprising modified nucleosides comprising at least two different sugar modifications. Unless otherwise indicated, a gapmer may comprise one or more modified internucleoside linkages and/or modified nucleobases and such modifications do not necessarily follow the gapmer pattern of the sugar modifications.

As used herein, “hotspot region” is a range of nucleobases on a target nucleic acid that is amenable to oligomeric compound-mediated reduction of the amount or activity of the target nucleic acid.

As used herein, “hybridization” means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, “internucleoside linkage” means the covalent linkage between contiguous nucleosides in an oligonucleotide. As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a phosphodiester internucleoside linkage. “Phosphorothioate internucleoside linkage or “PS internucleoside linkage” is a modified internucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester internucleoside linkage is replaced with a sulfur atom.

As used herein, “leukodystrophy” means a disorder due to abnormalities in the myelin sheath of neurons.

As used herein, “linker-nucleoside” means a nucleoside that links, either directly or indirectly, an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of an oligomeric compound. Linker-nucleosides are not considered part of the oligonucleotide portion of an oligomeric compound even if they are contiguous with the oligonucleotide.

As used herein, “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.

As used herein, “mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotide are aligned.

As used herein, “motif” means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.

As used herein, “nucleobase” means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase. A “5-methyl cytosine” is a modified nucleobase. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases. As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a target nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification.

As used herein, “nucleoside” means a compound, or a fragment of a compound, comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include a basic nucleosides, which lack a nucleobase. “Linked nucleosides” are nucleosides that are connected in a contiguous sequence (i.e., no additional nucleosides are presented between those that are linked).

As used herein, “oligomeric compound” means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A “singled-stranded oligomeric compound” is an unpaired oligomeric compound. The term “oligomeric duplex” means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a “duplexed oligomeric compound.”

As used herein, “oligonucleotide” means a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-50 linked nucleosides. As used herein, “modified oligonucleotide” means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.

As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to a subject. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffer solution or sterile artificial cerebrospinal fluid.

As used herein, “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

As used herein, “pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.

As used herein, “prodrug” means a therapeutic agent in a form outside the body that is converted to a different form within a subject or cells thereof. Typically, conversion of a prodrug within the subject is facilitated by the action of an enzymes (e.g., endogenous or viral enzyme) or chemicals present in cells or tissues and/or by physiologic conditions.

As used herein, “reducing the amount or activity” refers to a reduction or blockade of the transcriptional expression or activity relative to the transcriptional expression or activity in an untreated or control sample and does not necessarily indicate a total elimination of transcriptional expression or activity.

As used herein, “RNA” means an RNA transcript and includes pre-mRNA and mature mRNA unless otherwise specified.

As used herein, “RNAi compound” means an antisense compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics. In certain embodiments, an RNAi compound modulates the amount, activity, and/or splicing of a target nucleic acid. The term RNAi compound excludes antisense compounds that act through RNase H.

As used herein, “self-complementary” in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself.

As used herein, “standard in vitroassay” means the assay described in Example 1 and reasonable variations thereof.

As used herein, “standard in vivo assay” means the assay described in Example 7 and reasonable variations thereof.

As used herein, “stereorandom chiral center” in the context of a population of molecules of identical molecular formula means a chiral center having a random stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules having the (5) configuration of the stereorandom chiral center may be but is not necessarily the same as the number of molecules having the (R) configuration of the stereorandom chiral center. The stereochemical configuration of a chiral center is considered random when it is the result of a synthetic method that is not designed to control the stereochemical configuration. In certain embodiments, a stereorandom chiral center is a stereorandom phosphorothioate internucleoside linkage.

As used herein, “subject” means a human or non-human animal.

As used herein, “sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2′-OH(H) β-D-ribosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2′-H(H) β-D-deoxyribosyl sugar moiety, as found in DNA (an “unmodified DNA sugar moiety”). Unmodified sugar moieties have one hydrogen at each of the 1′, 3′, and 4′ positions, an oxygen at the 3′ position, and two hydrogens at the 5′ position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate.

As used herein, “sugar surrogate” means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or target nucleic acids.

As used herein, “symptom or hallmark” means any physical feature or test result that indicates the existence or extent of a disease or disorder. In certain embodiments, a symptom is apparent to a subject or to a medical professional examining or testing said subject. In certain embodiments, a hallmark is apparent upon invasive diagnostic testing, including, but not limited to, post-mortem tests. In certain embodiments, a hallmark is apparent on a brain MRI scan.

As used herein, “target nucleic acid” and “target RNA” mean a nucleic acid that an antisense compound is designed to affect.

As used herein, “target region” means a portion of a target nucleic acid to which an oligomeric compound is designed to hybridize.

As used herein, “terminal group” means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.

As used herein, “therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to a subject. For example, a therapeutically effective amount improves a symptom or hallmark of a disease.

Certain Embodiments

The present disclosure provides the following non-limiting numbered embodiments:

-   -   Embodiment 1: An oligomeric compound comprising a modified         oligonucleotide consisting of 12 to 30 linked nucleosides         wherein the nucleobase sequence of the modified oligonucleotide         is at least 90% complementary to an equal length portion of a         GFAP nucleic acid, and wherein the modified oligonucleotide         comprises at least one modification selected from a modified         sugar moiety and a modified internucleoside linkage.     -   Embodiment 2: An oligomeric compound comprising a modified         oligonucleotide consisting of 12 to 30 linked nucleosides and         having a nucleobase sequence comprising at least 12, at least         13, at least 14, at least 15, at least 16, at least 17, at least         18, at least 19, or 20 contiguous nucleobases of any of SEQ ID         NOs: 20-2809 or 2813, wherein the modified oligonucleotide         comprises at least one modification selected from a modified         sugar moiety and a modified internucleoside linkage.     -   Embodiment 3: An oligomeric compound comprising a modified         oligonucleotide consisting of 12 to 30 linked nucleosides and         having a nucleobase sequence comprising at least 12, at least         13, at least 14, at least 15, at least 16, at least 17, or 18         contiguous nucleobases of any of SEQ ID NOs: 2816-2837,         2839-2846, 2850-2854, 2856, 2859, 2861-2863, 2866, 2873-2876,         2886-2888, 2891, wherein the modified oligonucleotide comprises         at least one modification selected from a modified sugar moiety         and a modified internucleoside linkage.     -   Embodiment 4: An oligomeric compound comprising a modified         oligonucleotide consisting of 12 to 30 linked nucleosides and         having a nucleobase sequence comprising at least 8, at least 9,         at least 10, at least 11, at least 12, at least 13, at least 14,         at least 15, at least 16, at least 17, at least 18, at least 19,         or at least 20 contiguous nucleobases complementary to:         -   an equal length portion of nucleobases 9324-9348 of SEQ ID             NO: 2;         -   an equal length portion of nucleobases 9459-9480 of SEQ ID             NO: 2;         -   an equal length portion of nucleobases 9530-9580 of SEQ ID             NO: 2;         -   an equal length portion of nucleobases 12006-12038 of SEQ ID             NO: 2; or         -   an equal length portion of nucleobases 13038-13058 of SEQ ID             NO: 2,         -   wherein the modified oligonucleotide comprises at least one             modification selected from a modified sugar moiety and a             modified internucleoside linkage.     -   Embodiment 5: An oligomeric compound comprising a modified         oligonucleotide consisting of 12 to 30 linked nucleosides and         having a nucleobase sequence comprising at least 8, at least 9,         at least 10, at least 11, at least 12, at least 13, at least 14,         at least 15, at least 16, at least 17, or at least 18 contiguous         nucleobases of a sequence selected from:         -   SEQ ID Nos: 21, 1177, 2321, 2398, 2808-2809, 2840-2842,             2853-2854;         -   SEQ ID Nos: 555, 2093, 2170, 2813;         -   SEQ ID Nos: 20, 88, 166, 1331, 1408, 1485, 1637, 1713, 1714,             1789, 1790, 1637, 1638, 1865, 1866, 1941, 2018, 2095, 2172,             2249, 2326, 2403, 2480, 2557, 2633, 2709, 2785, 2816-2818,             2859, 2861, 2886-2887;         -   SEQ ID Nos: 815, 893, 971, 1049, 1269, 1270, 1346, 1423,             1499, 1500, 1660, 1736, 2655, 2731; or         -   SEQ ID Nos: 825, 1973.     -   Embodiment 6: The oligomeric compound of any of embodiments 1-5,         wherein the modified oligonucleotide has a nucleobase sequence         that is at least 80%, at least 85%, at least 90%, at least 95%,         or 100% complementary to the nucleobase sequence of SEQ ID NO:         1, SEQ ID NO: 2 or SEQ ID NO: 3 when measured across the entire         nucleobase sequence of the modified oligonucleotide.     -   Embodiment 7: The oligomeric compound of any of embodiments 1-6,         wherein the modified oligonucleotide comprises at least one         modified sugar moiety.     -   Embodiment 8: The oligomeric compound of embodiment 7, wherein         the modified oligonucleotide comprises at least one bicyclic         sugar moiety.     -   Embodiment 9: The oligomeric compound of embodiment 8, wherein         the bicyclic sugar moiety has a 4′-2′ bridge, wherein the 4′-2′         bridge is selected from CH₂—O—; and —CH(CH₃)—O.     -   Embodiment 10: The oligomeric compound of any of embodiments         1-9, wherein the modified oligonucleotide comprises at least one         non-bicyclic modified sugar moiety.     -   Embodiment 11: The oligomeric compound of embodiment 10, wherein         the non-bicyclic modified sugar moiety is a 2′-MOE sugar moiety         or a 2′-OMe sugar moiety.     -   Embodiment 12: The oligomeric compound of any of embodiments         1-11, wherein the modified oligonucleotide comprises at least         one sugar surrogate.     -   Embodiment 13: The oligomeric compound of embodiment 12, wherein         the sugar surrogate is any of morpholino, modified morpholino,         PNA, THP, and F-HNA.     -   Embodiment 14: The oligomeric compound of any of embodiments 1-7         or 10-13, wherein the modified oligonucleotide does not comprise         a bicyclic sugar moiety.     -   Embodiment 15: The oligomeric compound of any of embodiments         1-14, wherein the modified oligonucleotide is a gapmer.     -   Embodiment 16: The oligomeric compound of any of embodiments         1-15, wherein the modified oligonucleotide comprises:         -   a 5′-region consisting of 1-6 linked nucleosides;         -   a central region consisting of 6-10 linked nucleosides; and         -   a 3′-region consisting of 1-6 linked nucleosides; wherein             each of the 5′-region nucleosides and each of the 3′-region             nucleosides comprises a modified sugar moiety and at least             one of the central region nucleosides comprises a             2′-deoxyribosyl sugar moiety.     -   Embodiment 17: The oligomeric compound of embodiment 16, wherein         the modified oligonucleotide comprises         -   a 5′-region consisting of 6 linked nucleosides;         -   a central region consisting of 10 linked nucleosides; and         -   a 3′-region consisting of 4 linked nucleosides; wherein         -   each of the 5′-region nucleosides and each of the 3′-region             nucleosides comprises a 2′-MOE modified sugar moiety, and             each of the central region nucleosides comprises a             2′-deoxyribosyl sugar moiety.     -   Embodiment 18: The oligomeric compound of embodiment 16, wherein         the modified oligonucleotide comprises:         -   a 5′-region consisting of 5 linked nucleosides;         -   a central region consisting of 10 linked nucleosides; and         -   a 3′-region consisting of 5 linked nucleosides; wherein         -   each of the 5′-region nucleosides and each of the 3′-region             nucleosides comprises a 2′-MOE modified sugar moiety, and             each of the central region nucleosides comprises a             2′-deoxyribosyl sugar moiety.     -   Embodiment 19: The oligomeric compound of embodiment 16, wherein         the modified oligonucleotide comprises:         -   a 5′-region consisting of 4 linked nucleosides;         -   a central region consisting of 10 linked nucleosides; and         -   a 3′-region consisting of 6 linked nucleosides; wherein         -   each of the 5′-region nucleosides and each of the 3′-region             nucleosides comprises a 2′-MOE modified sugar moiety, and             each of the central region nucleosides comprises a             2′-deoxyribosyl sugar moiety.     -   Embodiment 20: The oligomeric compound of embodiment 16, wherein         the modified oligonucleotide comprises         -   a 5′-region consisting of 5 linked nucleosides;         -   a central region consisting of 8 linked nucleosides; and         -   a 3′-region consisting of 5 linked nucleosides; wherein         -   each of the 5′-region nucleosides and each of the 3′-region             nucleosides comprises a 2′-MOE modified sugar moiety, and             each of the central region nucleosides comprises a             2′-deoxyribosyl sugar moiety.     -   Embodiment 21: The oligomeric compound of any of embodiments         1-20, wherein the modified oligonucleotide comprises at least         one modified internucleoside linkage.     -   Embodiment 22: The oligomeric compound of embodiment 21, wherein         each internucleoside linkage of the modified oligonucleotide is         a modified internucleoside linkage.     -   Embodiment 23: The oligomeric compound of embodiments 21 or 22,         wherein the modified internucleoside linkage is a         phosphorothioate internucleoside linkage.     -   Embodiment 24: The oligomeric compound of any one of embodiments         1-21, wherein the modified oligonucleotide comprises at least         one phosphodiester internucleoside linkage.     -   Embodiment 25: The oligomeric compound of any of embodiments 21,         23, or 24, wherein each internucleoside linkage is either a         phosphodiester internucleoside linkage or a phosphorothioate         internucleoside linkage.     -   Embodiment 26: The oligomeric compound of any one of embodiments         1-21 or 23-25, wherein the modified oligonucleotide has an         internucleoside linkage motif selected from among:         sooosssssssssssooss, sooooossssssssssoss, soooossssssssssooss,         sooosssssssssooss, or sooossssssssssoooss; wherein, s=a         phosphorothioate internucleoside linkage and o=a phosphodiester         internucleoside linkage.     -   Embodiment 27: The oligomeric compound of any of embodiments         1-26, wherein the modified oligonucleotide comprises at least         one modified nucleobase.     -   Embodiment 28: The oligomeric compound of embodiment 27, wherein         the modified nucleobase is a 5-methylcytosine.     -   Embodiment 29: The oligomeric compound of any of embodiments         1-28, wherein the modified oligonucleotide consists of 12-30,         12-22, 12-20, 14-18, 14-20, 15-17, 15-25, 16-20, 18-22 or 18-20         linked nucleosides.     -   Embodiment 30: The oligomeric compound of any of embodiments         1-29, wherein the modified oligonucleotide consists of 18 linked         nucleosides.     -   Embodiment 31: The oligomeric compound of any of embodiments         1-30, wherein the modified oligonucleotide consists of 20 linked         nucleosides.     -   Embodiment 32: The oligomeric compound of any of embodiments         1-31, consisting of the modified oligonucleotide.     -   Embodiment 33: An oligomeric duplex comprising an oligomeric         compound of any of embodiments 1-31.     -   Embodiment 34: An antisense compound comprising or consisting of         an oligomeric compound of any of embodiments 1-32 or an         oligomeric duplex of embodiment 33.     -   Embodiment 35: A pharmaceutical composition comprising an         oligomeric compound of any of embodiments 1-32 or an oligomeric         duplex of embodiment 33 and a pharmaceutically acceptable         carrier or diluent.     -   Embodiment 36: The pharmaceutical composition of embodiment 35,         wherein the pharmaceutically acceptable diluent is artificial         cerebral spinal fluid.     -   Embodiment 37: The pharmaceutical composition of embodiment 36,         wherein the pharmaceutical composition consists of the modified         oligonucleotide and phosphate buffered saline.     -   Embodiment 38: A method comprising administering to a subject a         pharmaceutical composition of any of embodiments 35-37.     -   Embodiment 39: A method of treating a disease associated with         GFAP comprising administering to an individual having or at risk         for developing a disease associated with GFAP a therapeutically         effective amount of a compound of any one of embodiments 1-34 or         a pharmaceutical composition according to any of embodiments         35-37; and thereby treating the disease associated with GFAP.     -   Embodiment 40: The method of embodiment 39, wherein the         GFAP-associated disease is Alexander Disease.     -   Embodiment 41: The method of any of embodiments 38-40, wherein         at least one symptom or hallmark of the GFAP-associated disease         is ameliorated.     -   Embodiment 42: The method of embodiment 41, wherein the symptom         or hallmark is motor delays, cognitive delays, paroxysmal         deterioration, seizures, vomiting, swallowing difficulties,         ataxic gait, palatal myoclonus, autonomic dysfunction, or the         presence of intra-astrocytic inclusions called Rosenthal fibers.     -   Embodiment 43: The method of any of embodiments 38-42, wherein         GFAP levels in the individual are reduced.     -   Embodiment 44: A modified oligonucleotide according to the         following chemical structure:

or a salt thereof.

-   -   Embodiment 45: A modified oligonucleotide according to the         following chemical structure:

-   -   Embodiment 46: A modified oligonucleotide according to the         following chemical structure:

or a salt thereof.

-   -   Embodiment 47: A modified oligonucleotide according to the         following chemical structure:

-   -   Embodiment 48: A modified oligonucleotide according to the         following chemical structure:

or a salt thereof.

-   -   Embodiment 49: A modified oligonucleotide according to the         following chemical structure:

-   -   Embodiment 50: A modified oligonucleotide according to the         following chemical structure:

or a salt thereof

-   -   Embodiment 51: A modified oligonucleotide according to the         following chemical structure:

-   -   Embodiment 52: A modified oligonucleotide according to the         following chemical structure:

or a salt thereof

-   -   Embodiment 53: A modified oligonucleotide according to the         following chemical structure:

-   -   Embodiment 54: A modified oligonucleotide according to the         following chemical structure:

or a salt thereof

-   -   Embodiment 55: A modified oligonucleotide according to the         following chemical structure:

-   -   Embodiment 56: The modified oligonucleotide of any one of         embodiments 44, 46, 48, 50, 52, and 54, which is the sodium salt         or potassium salt of the chemical structure.     -   Embodiment 57: A pharmaceutical composition comprising the         modified oligonucleotide of any of embodiments 44-56 and a         pharmaceutically acceptable carrier or diluent.     -   Embodiment 58: The pharmaceutical composition of embodiment 57,         wherein the pharmaceutically acceptable diluent is artificial         cerebrospinal fluid.     -   Embodiment 59: The pharmaceutical composition of embodiment 58,         wherein the pharmaceutical composition consists of the modified         oligonucleotide and artificial cerebrospinal fluid.     -   Embodiment 60: A compound comprising a modified oligonucleotide         according to the following chemical notation:         ^(m)C_(es)A_(eo)G_(eo)T_(eo)A_(eo)T_(eo)T_(ds)A_(ds) ^(m)C_(ds)         ^(m)C_(ds)T_(ds) ^(m)C_(ds)T_(ds)A_(ds)         ^(m)C_(ds)T_(ds)A_(eo)G_(es)T_(es) ^(m)C_(es) (SEQ ID NO: 20),         wherein:     -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.     -   Embodiment 61: A compound comprising a modified oligonucleotide         according to the following chemical notation: ^(m)C_(es)A_(eo)         ^(m)C_(eo)A_(eo)T_(eo)T_(eo) ^(m)C_(ds)A_(ds)         ^(m)C_(ds)T_(ds)A_(ds)A_(ds)T_(ds)A_(ds)T_(ds)T_(ds)T_(eo)A_(es)A_(es)         ^(m)C_(es) (SEQ ID NO: 21), wherein:     -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.     -   Embodiment 62: A compound comprising a modified oligonucleotide         according to the following chemical notation: ^(m)C_(es)         ^(m)C_(eo)A_(eo)G_(eo)T_(eo)G_(ds)T_(ds) ^(m)C_(ds)T_(ds)T_(ds)         ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(eo)G_(eo)         ^(m)C_(es)T_(es) ^(m)C_(e) (SEQ ID NO: 825), wherein:     -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.     -   Embodiment 63: A compound comprising a modified oligonucleotide         according to the following chemical notation: G_(es)         ^(m)C_(eo)A_(eo)A_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds)T_(ds)T_(ds)         ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)T_(ds)A_(ds)A_(eo)         ^(m)C_(eo)A_(es)A_(es) ^(m)C_(e) (SEQ ID NO: 1499), wherein:     -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.     -   Embodiment 64: A compound comprising a modified oligonucleotide         according to the following chemical notation:         T_(es)G_(eo)G_(eo)T_(eo) ^(m)C_(eo)         ^(m)C_(ds)T_(ds)A_(ds)A_(ds)A_(ds)T_(ds)A_(ds)T_(ds)T_(ds)         ^(m)C_(ds)T_(eo)A_(eo)G_(es)T_(es) ^(m)C_(e) (SEQ ID NO: 2170),         wherein:     -   A=an adenine nucleobase,     -   m^(C)=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.     -   Embodiment 65: A compound comprising a modified oligonucleotide         according to the following chemical notation: T_(es)G_(eo)G_(eo)         ^(m)C_(eo)A_(es)G_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)         ^(m)C_(ds)T_(eo) ^(m)C_(eo)T_(es)A_(es) ^(m)C_(e) (SEQ ID NO:         2818), wherein:     -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.     -   Embodiment 66: The compound of any of embodiments 60-65,         comprising the modified oligonucleotide covalently linked to a         conjugate group.     -   Embodiment 67: A pharmaceutical composition comprising a         compound of any of embodiments 60-66, and a pharmaceutically         acceptable diluent or carrier.     -   Embodiment 68: The pharmaceutical composition of embodiment 67,         wherein the pharmaceutically acceptable diluent is artificial         cerebrospinal fluid.     -   Embodiment 69: The pharmaceutical composition of embodiment 68,         wherein the pharmaceutical composition consists of the compound         and artificial cerebrospinal fluid.     -   Embodiment 70: A chirally enriched population of modified         oligonucleotides of any of embodiments 60-65, wherein the         population is enriched for modified oligonucleotides comprising         at least one particular phosphorothioate internucleoside linkage         having a particular stereochemical configuration.     -   Embodiment 71: The chirally enriched population of embodiment         70, wherein the population is enriched for modified         oligonucleotides comprising at least one particular         phosphorothioate internucleoside linkage having the (Sp)         configuration.     -   Embodiment 72: The chirally enriched population of embodiment         70, wherein the population is enriched for modified         oligonucleotides comprising at least one particular         phosphorothioate internucleoside linkage having the (Rp)         configuration.     -   Embodiment 73: The chirally enriched population of embodiment         70, wherein the population is enriched for modified         oligonucleotides having a particular, independently selected         stereochemical configuration at each phosphorothioate         internucleoside linkage     -   Embodiment 74: The chirally enriched population of embodiment         70, wherein the population is enriched for modified         oligonucleotides having the (Sp) configuration at each         phosphorothioate internucleoside linkage.     -   Embodiment 75: The chirally enriched population of embodiment         70, wherein the population is enriched for modified         oligonucleotides having the (Rp) configuration at each         phosphorothioate internucleoside linkage.     -   Embodiment 76: The chirally enriched population of embodiment         70, wherein the population is enriched for modified         oligonucleotides having the (Rp) configuration at one particular         phosphorothioate internucleoside linkage and the (Sp)         configuration at each of the remaining phosphorothioate         internucleoside linkages.     -   Embodiment 77: The chirally enriched population of embodiment 70         or embodiment 73 wherein the population is enriched for modified         oligonucleotides having at least 3 contiguous phosphorothioate         internucleoside linkages in the Sp, Sp, and Rp configurations,         in the 5′ to 3′ direction.     -   Embodiment 78: A chirally enriched population of modified         oligonucleotides of any of embodiments 44-55, wherein all of the         phosphorothioate internucleoside linkages of the modified         oligonucleotide are stereorandom.     -   Embodiment 79: A method comprising administering to an         individual the pharmaceutical composition of any of embodiments         56-58 and 67-69.     -   Embodiment 80: A method of treating a disease associated with         GFAP, comprising administering to an individual having or at         risk of having a disease associated with GFAP a therapeutically         effective amount of the pharmaceutical composition of any one of         embodiments 56-58 and 67-69, thereby treating the disease         associated with GFAP.     -   Embodiment 81: A method of reducing GFAP protein in the CSF of         an individual having having or at risk of having a disease         associated with GFAP a therapeutically effective amount of a         pharmaceutical composition of any one of embodiments 56-58 and         67-69, thereby reducing GFAP protein in the CSF.     -   Embodiment 82: The method of embodiment 80 or embodiment 81,         wherein the disease is a neurodegenerative disease.     -   Embodiment 83: The method of any of embodiments 80-82, wherein         the disease is Alexander disease.     -   Embodiment 84: The method of any of embodiments 80-83, wherein         at least one symptom or hallmark of the disease is ameliorated.     -   Embodiment 85: The method of embodiment 84, wherein the symptom         or hallmark is any of motor delays, cognitive delays, paroxysmal         deterioration, seizures, vomiting, swallowing difficulties,         ataxic gait, palatal myoclonus, autonomic dysfunction, or the         presence of intra-astrocytic inclusions called Rosenthal fibers.     -   Embodiment 86: The method of any of embodiments 79-85, wherein         the pharmaceutical composition is administered to the central         nervous system or systemically.     -   Embodiment 87: The method of embodiment 86, wherein wherein the         pharmaceutical composition is administered to the central         nervous system and systemically.     -   Embodiment 88: The method of any of embodiments 80-87, wherein         the pharmaceutical composition is administered any of         intrathecally, systemically, subcutaneously, or intramuscularly.

I. Certain Oligonucleotides

In certain embodiments, provided herein are oligomeric compounds comprising oligonucleotides, which consist of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA. That is, modified oligonucleotides comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage.

A. Certain Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

1. Certain Sugar Moieties

In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.

In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups none of which bridges two atoms of the fumnosyl ring to form a bicyclic structure. Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2′, 4′, and/or 5′ positions. In certain embodiments one or more non-bridging substituent of non-bicyclic modified sugar moieties is branched. Examples of 2′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE” or “O-methoxyethyl”). In certain embodiments, 2′-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃, O—C₁-C₁₀ alkoxy, O—C₁-C₁₀ substituted alkoxy, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl, S-alkyl, N(R_(m))-alkyl, O-alkenyl, S-alkenyl, N(R_(m))-alkenyl, O-alkynyl, S-alkynyl, N(R_(m))-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O(CH₂)₂ON(R_(m))(R_(n)) or OCH₂C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀ alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat. No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al., U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128. Examples of 5′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certain embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.

In certain embodiments, a 2′-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2′-substituent group selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂, CH₂CH═CH₂, OCH₂CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂O N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide (OCH₂C(═O)—N(R_(m))(R_(n))), where each R_(m) and R_(n) is, independently, H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2′-substituent group selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, and OCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2′-substituent group selected from: F, OCH₃, and OCH₂CH₂OCH₃.

In certain embodiments, modified furanosyl sugar moieties and nucleosides incorporating such modified furanosyl sugar moieties are further defined by isomeric configuration. For example, a 2′-deoxyfuranosyl sugar moiety may be in seven isomeric configurations other than the naturally occurring β-D-deoxyribosyl configuration. Such modified sugar moieties are described in, e.g., WO 2019/157531, incorporated by reference herein. A 2′-modified sugar moiety has an additional stereocenter at the 2′-position relative to a 2′-deoxyfuranosyl sugar moiety; therefore, such sugar moieties have a total of sixteen possible isomeric configurations. 2′-modified sugar moieties described herein are in the β-D-ribosyl isomeric configuration unless otherwise specified.

Certain modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ bridging sugar substituents include but are not limited to: 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′ (“LNA”), 4′-CH₂—S-2′, 4′-(CH₂)₂—O-2′ (“ENA”), 4′-CH(CH₃)—O-2′ (referred to as “constrained ethyl” or “cEt”), 4′-CH₂—N(R)-2′, 4′-CH(CH₂OCH₃)—O-2′ (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No. 8,022,193), 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4′-CH₂—O—N(CH₃)-2′ (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see e.g., Seth et al., U.S. Pat. No. 8,278,426), 4′-C(R_(a)R_(b))—N(R)—O-2′, 4′-C(R_(a)R_(b))—O—N(R)-2′, 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O- 2′, wherein each R, R_(a), and R_(b) is, independently, H, a protecting group, or C₁-C₁₂ alkyl (see, e.g. Imanishi et al., U.S. Pat. No. 7,427,672).

In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from: —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))_(z)—, —S(═O)_(x)—, and —N(R_(a))—;

-   -   wherein:     -   x is 0, 1, or 2;     -   n is 1, 2, 3, or 4;     -   each R_(a) and R_(b) is, independently, H, a protecting group,         hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂         alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted         C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,         heterocycle radical, substituted heterocycle radical,         heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,         substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,         N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl         (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and     -   each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted         C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂         alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted         C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle         radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,         substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129, 8362-8379; Wengel et a., U.S. Pat. No. 7,053,207; Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al. U.S. Pat. No. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al., U.S. Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengel et al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; Ramasamy et al., U.S. Pat. No. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; and U.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawa et al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the a-L configuration or in the β-D configuration.

α-L-methyleneoxy (4′-CH₂—O-2′) or a-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars).

In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No. 7,939,677) and/or the 5′ position.

In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”) (see, e.g., Leumann, C J. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:

(“F-HNA”, see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze et al., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No. 8,796,437; and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can also be referred to as a F-THP or 3′-fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula:

wherein, independently, for each of the modified THP nucleosides:

-   -   Bx is a nucleobase moiety;     -   T₃ and T₄ are each, independently, an internucleoside linking         group linking the modified THP nucleoside to the remainder of an         oligonucleotide or one of T₃ and T₄ is an internucleoside         linking group linking the modified THP nucleoside to the         remainder of an oligonucleotide and the other of T₃ and T₄ is H,         a hydroxyl protecting group, a linked conjugate group, or a 5′         or 3′-terminal group;         q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆         alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆         alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl; and     -   each of R₁ and R₂ is independently selected from among:         hydrogen, halogen, substituted or unsubstituted alkoxy, NJ₁J₂,         SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X         is O, S or NJ₁, and each J₁, J₂, and J₃ is, independently, H or         C₁-C₆ alkyl.

In certain embodiments, modified THP nucleosides are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, modified THP nucleosides are provided wherein one of R1 and R₂ is F. In certain embodiments, R₁ is F and R₂ is H, in certain embodiments, R₁ is methoxy and R₂ is H, and in certain embodiments, R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

2. Certain Modified Nucleobases

In certain embodiments, modified oligonucleotides comprise one or more nucleosides comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an a basic nucleoside.

In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (—C≡C—CH₃) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp) Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.

Publications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, Manoharan et al., US2003/0158403; Manoharan et al., US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al., U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066; Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat. No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al., U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cook et al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No. 5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al., U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540; Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No. 5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S. Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook et al., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cook et al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903; Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No. 5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al., U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook et al., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No. 6,005,096.

3. Certain Modified Internucleoside Linkages

In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphodiesters, which contain a phosphodiester bond (“P(O₂)═O”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, phosphorothioates (“P(O₂)═S”), and phosphorodithioates (“HS—P═S”). Representative non-phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester, thionocarbamate (—O—C(═O)(NH)—S—); siloxane (—O—SiH₂—O—); and N,N-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Modified internucleoside linkages, compared to naturally occurring phosphodiester internucleoside linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

Representative internucleoside linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom internucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate internucleoside linkages in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise phosphorothioate internucleoside linkages wherein all of the phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate internucleoside linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate internucleoside linkage in a particular, independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate internucleoside linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO 2017/015555. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (Sp) phosphorothioates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:

Unless otherwise indicated, chiral internucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.

Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3 (3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal (3′-O—CH₂—O-5′), methoxypropyl (MOP), and thioformacetal (3′-S—CH₂—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (see for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH₂ component parts.

B. Certain Motifs

In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).

1. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or portion thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides have a gapmer motif, which is defined by two external regions or “wings” and a central or internal region or “gap.” The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5′-wing differs from the sugar motif of the 3′-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-6 nucleosides. In certain embodiments, each nucleoside of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least one nucleoside of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least two nucleosides of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least three nucleosides of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least four nucleosides of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, at least five nucleosides of each wing of a gapmer comprises a modified sugar moiety.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer comprises a 2-deoxyribosyl sugar moiety. In certain embodiments, each nucleoside of the gap of a gapmer comprises a 2′-β-D-deoxyribosyl sugar moiety. In certain embodiments, at least one nucleoside of the gap of a gapmer comprises a modified sugar moiety. In certain embodiments, at least one nucleoside of the gap of a gapmer comprises a 2′-OMe sugar moiety.

In certain embodiments, the gapmer is a deoxy gapmer. In certain embodiments, the nucleosides on the gap side of each wing/gap junction comprise 2′-deoxyribosyl sugar moieties and the nucleosides on the wing sides of each wing/gap junction comprise modified sugar moieties. In certain embodiments, each nucleoside of the gap comprises a 2-deoxyribosyl sugar moiety. In certain embodiments, each nucleoside of each wing of a gapmer comprises a modified sugar moiety. In certain embodiments, one nucleoside of the gap comprises a modified sugar moiety and each remaining nucleoside of the gap comprises a 2-deoxyribosyl sugar moiety.

In certain embodiments, modified oligonucleotides comprise or consist of a portion having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified portion of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a portion having a fully modified sugar motif, wherein each nucleoside within the fully modified portion comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified oligonucleotide comprises the same 2′-modification.

Herein, the lengths (number of nucleosides) of the three regions of a gapmer may be provided using the notation [# of nucleosides in the 5′-wing]−[# of nucleosides in the gap]−[# of nucleosides in the 3′-wing]. Thus, a 5-10-5 gapmer consists of 5 linked nucleosides in each wing and 10 linked nucleosides in the gap. Where such nomenclature is followed by a specific modification, that modification is the modification in each sugar moiety of each wing and the gap nucleosides comprises a 2′-β-D-deoxyribosyl sugar moiety. Thus, a 5-10-5 MOE gapmer consists of 5 linked 2′-MOE nucleosides in the 5′-wing, 10 linked 2′-β-D-deoxynucleosides in the gap, and 5 linked 2′-MOE nucleosides in the 3′-wing. A 3-10-3 cEt gapmer consists of 3 linked cEt nucleosides in the 5′-wing, 10 linked 2′-β-D-deoxynucleosides in the gap, and 3 linked cEt nucleosides in the 3′-wing. A 5-8-5 gapmer consists of 5 linked nucleosides comprising a modified sugar moiety in the 5′-wing, 8 linked 2′-deoxynucleosides in the gap, and 5 linked nucleosides comprising a modified sugar moiety in the 3′-wing. A mixed wing gapmer has at least two different modified sugars in the 5′ and/or 3′ wing. A 5-8-5 or 5-8-4 mixed wing gapmer has at least two different modified sugar moieties in the 5′- and/or the 3′-wing.

In certain embodiments, modified oligonucleotides are 5-10-5 MOE gapmers. In certain embodiments, modified oligonucleotides are 4-10-6 MOE gapmers. In certain embodiments, modified oligonucleotides are 6-10-4 MOE gapmers. In certain embodiments, modified oligonucleotides are 5-8-5 MOE gapmers. In certain embodiments, modified oligonucleotides are X—Y—Z MOE gapmers, wherein X and Z are independently selected from 1, 2, 3, 4, 5, or 6 and Y is 7, 8, 9, 10, or 11.

In certain embodiments, modified oligonucleotides have the following sugar motif (5′ to 3′): meeemddddddddddmmmmm, wherein ‘d’ represents a 2′-deoxyribosyl sugar moiety, ‘e’ represents a 2′-MOE sugar moiety, and ‘m’ represents a 2′-OMe sugar moiety.

2. Certain Nucleobase Motifs

In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or portion thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methyl cytosines. In certain embodiments, all of the cytosine nucleobases are 5-methyl cytosines and all of the other nucleobases of the modified oligonucleotide are unmodified nucleobases.

In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 3′-end of the oligonucleotide. In certain embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5′-end of the oligonucleotide.

In certain embodiments, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of the nucleoside is a 2′-deoxyribosyl sugar moiety. In certain embodiments, the modified nucleobase is selected from: a 2-thiopyrimidine and a 5-propynepyrimidine.

3. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each internucleoside linking group is a phosphodiester internucleoside linkage (P═O). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate internucleoside linkage (P═S). In certain embodiments, each internucleoside linkage of a modified oligonucleotide is independently selected from a phosphorothioate internucleoside linkage and phosphodiester internucleoside linkage. In certain embodiments, each phosphorothioate internucleoside linkage is independently selected from a stereorandom phosphorothioate, a (Sp) phosphorothioate, and a (Rp) phosphorothioate. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap are all modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphodiester internucleoside linkages. In certain embodiments, the terminal internucleoside linkages are modified. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer, and the internucleoside linkage motif comprises at least one phosphodiester internucleoside linkage in at least one wing, wherein the at least one phosphodiester internucleoside linkage is not a terminal internucleoside linkage, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. In certain such embodiments, all of the phosphorothioate internucleoside linkages are stereorandom. In certain embodiments, all of the phosphorothioate internucleoside linkages in the wings are (Sp) phosphorothioates, and the gap comprises at least one Sp, Sp, Rp motif. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such internucleoside linkage motifs.

In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of sooosssssssssssooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of (5′ to 3′): sooooossssssssssoss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of (5′ to 3′): soooossssssssssooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of (5′ to 3′):sooosssssssssooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of (5′ to 3′): sooossssssssssoooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. In certain embodiments, modified oligonucleotides have an internucleoside linkage motif of (5′ to 3′): sooosssssssssssssss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage.

C. Certain Lengths

It is possible to increase or decrease the length of an oligonucleotide without eliminating activity. For example, in Woolf et al. Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992) a series of oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target nucleic acid in an oocyte injection model. Oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the oligonucleotides were able to direct specific cleavage of the target nucleic acid, albeit to a lesser extent than the oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase oligonucleotides, including those with 1 or 3 mismatches.

In certain embodiments, oligonucleotides (including modified oligonucleotides) can have any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, in certain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides.

D. Certain Modified Oligonucleotides

In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification motifs and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif. Likewise, such sugar gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Unless otherwise indicated, all modifications are independent of nucleobase sequence.

E. Certain Populations of Modified Oligonucleotides

Populations of modified oligonucleotides in which all of the modified oligonucleotides of the population have the same molecular formula can be stereorandom populations or chirally enriched populations. All of the chiral centers of all of the modified oligonucleotides are stereorandom in a stereorandom population. In a chirally enriched population, at least one particular chiral center is not stereorandom in the modified oligonucleotides of the population. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for β-D ribosyl sugar moieties, and all of the phosphorothioate internucleoside linkages are stereorandom. In certain embodiments, the modified oligonucleotides of a chirally enriched population are enriched for both β-D ribosyl sugar moieties and at least one, particular phosphorothioate internucleoside linkage in a particular stereochemical configuration.

F. Nucleobase Sequence

In certain embodiments, oligonucleotides (unmodified or modified oligonucleotides) are further described by their nucleobase sequence. In certain embodiments oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain such embodiments, a portion of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid. In certain embodiments, the nucleobase sequence of a portion or entire length of an oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.

II. Certain Oligomeric Compounds

In certain embodiments, provided herein are oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2′-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3′ and/or 5′-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3′-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5′-end of oligonucleotides.

Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, a basic nucleosides, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

A. Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide. Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/179620).

In certain embodiments, conjugate groups may be selected from any of a C22 alkyl, C20 alkyl, C16 alkyl, C10 alkyl, C21 alkyl, C19 alkyl, C18 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, C11 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl, C22 alkenyl, C20 alkenyl, C16 alkenyl, C10 alkenyl, C21 alkenyl, C19 alkenyl, C18 alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, C11 alkenyl, C9 alkenyl, C8 alkenyl, C7 alkenyl, C6 alkenyl, or C5 alkenyl.

In certain embodiments, conjugate groups may be selected from any of C22 alkyl, C20 alkyl, C16 alkyl, C10 alkyl, C21 alkyl, C19 alkyl, C18 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, C11 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, and C5 alkyl, where the alkyl chain has one or more unsaturated bonds.

1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates, vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, lipophilic groups, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.

In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

2. Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain oligomeric compounds, the conjugate linker is a single chemical bond (i.e., the conjugate moiety is attached directly to an oligonucleotide through a single bond). In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a parent compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-calboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides. In certain embodiments, conjugate linkers comprise 2-5 linker-nucleosides. In certain embodiments, conjugate linkers comprise exactly 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise the TCA motif. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methyl cytosine, 4-N-benzoyl-5-methyl cytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which an oligomeric compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the oligomeric compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, an oligomeric compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such an oligomeric compound is more than 30. Alternatively, an oligomeric compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such an oligomeric compound is no more than 30. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances oligomeric compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the oligomeric compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate linkers may comprise one or more cleavable moieties. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate or phosphodiester internucleoside linkage between an oligonucleotide and a conjugate moiety or conjugate group.

In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, the one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2′-deoxynucleoside that is attached to either the 3′ or 5′-terminal nucleoside of an oligonucleotide by a phosphodiester internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate internucleoside linkage. In certain such embodiments, the cleavable moiety is 2′-deoxyadenosine.

3. Cell-Targeting Moieties

In certain embodiments, a conjugate group comprises a cell-targeting moiety. In certain embodiments, a conjugate group has the general formula:

wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2 or greater, j is 1 or 0, and k is 1 or 0.

In certain embodiments, n is 1, j is 1 and k is 0. In certain embodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is 0 and k is 1. In certain embodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1.

In certain embodiments, conjugate groups comprise cell-targeting moieties that have at least one tethered ligand. In certain embodiments, cell-targeting moieties comprise two tethered ligands covalently attached to a branching group. In certain embodiments, cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.

B. Certain Terminal Groups

In certain embodiments, oligomeric compounds comprise one or more terminal groups. In certain such embodiments, oligomeric compounds comprise a stabilized 5′-phosphate. Stabilized 5′-phosphates include, but are not limited to 5′-phosphonates, including, but not limited to 5′-vinylphosphonates. In certain embodiments, terminal groups comprise one or more a basic nucleosides and/or inverted nucleosides. In certain embodiments, terminal groups comprise one or more 2′-linked nucleosides. In certain such embodiments, the 2′-linked nucleoside is an a basic nucleoside.

III. Oligomeric Duplexes

In certain embodiments, oligomeric compounds described herein comprise an oligonucleotide, having a nucleobase sequence complementary to that of a target nucleic acid. In certain embodiments, an oligomeric compound is paired with a second oligomeric compound to form an oligomeric duplex. Such oligomeric duplexes comprise a first oligomeric compound having a portion complementary to a target nucleic acid and a second oligomeric compound having a portion complementary to the first oligomeric compound. In certain embodiments, the first oligomeric compound of an oligomeric duplex comprises or consists of (1) a modified or unmodified oligonucleotide and optionally a conjugate group and (2) a second modified or unmodified oligonucleotide and optionally a conjugate group. Either or both oligomeric compounds of an oligomeric duplex may comprise a conjugate group. The oligonucleotides of each oligomeric compound of an oligomeric duplex may include non-complementary overhanging nucleosides.

IV. Antisense Activity

In certain embodiments, oligomeric compounds and oligomeric duplexes are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity; such oligomeric compounds and oligomeric duplexes are antisense compounds. In certain embodiments, antisense compounds have antisense activity when they reduce the amount or activity of a target nucleic acid by 25% or more in the standard cell assay. In certain embodiments, antisense compounds selectively affect one or more target nucleic acid. Such antisense compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in significant undesired antisense activity.

In certain antisense activities, hybridization of an antisense compound to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, described herein are antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. In certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.

In certain antisense activities, an antisense compound or a portion of an antisense compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain antisense compounds result in cleavage of the target nucleic acid by Argonaute Antisense compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA) or single-stranded (ssRNA).

In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain embodiments, hybridization of the antisense compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid results in alteration of translation of the target nucleic acid.

Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein and/or a phenotypic change in a cell or subject.

V. Certain Target Nucleic Acids

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide comprising a portion that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: a mature mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target nucleic acid is a mature mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.

A. Complementarity/Mismatches to the Target Nucleic Acid

It is possible to introduce mismatch bases without eliminating activity. For example, Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase oligonucleotides, and a 28- and 42-nucleobase oligonucleotides comprised of the sequence of two or three of the tandem oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase oligonucleotides.

In certain embodiments, oligonucleotides are complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99%, 95%, 90%, 85%, or 80% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a portion that is 100% or fully complementary to a target nucleic acid. In certain embodiments, the portion of full complementarity is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleobases in length.

In certain embodiments, oligonucleotides comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain embodiments selectivity of the oligonucleotide is improved. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 from the 5′-end of the gap region. In certain embodiments, the mismatch is at position 1, 2, 3, 4, 5, or 6 from the 5′-end of the 5′ wing region or the 3′ wing region.

B. GFAP

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide that is complementary to a target nucleic acid, wherein the target nucleic acid is a GFAP nucleic acid. In certain embodiments, GFAP nucleic acid has the sequence set forth in SEQ ID NO: 1 (GENBANK Accession No. NM_002055.4), SEQ ID NO: 2 (GENBANK Accession No. NC_000017.11 truncated from nucleotides 44903001 to 44919000), or SEQ ID NO: 3 (GENBANK Accession No. NM_001131019.2).

In certain embodiments, contacting a cell with an oligomeric compound complementary to any of SEQ ID NO: 1-3 reduces the amount of GFAP RNA and in certain embodiments reduces the amount of GFAP protein. In certain embodiments, the oligomeric compound consists of a modified oligonucleotide. In certain embodiments, contacting a cell with an oligomeric compound complementary to any of SEQ ID NO: 1-3 reduces the amount of GFAP RNA in a cell, and in certain embodiments reduces the amount of GFAP protein in a cell. In certain embodiments, the cell is in vitro. In certain embodiments, the cell is in a subject. In certain embodiments, the oligomeric compound consists of a modified oligonucleotide. In certain embodiments, contacting a cell in a subject with an oligomeric compound complementary to any of SEQ ID NO: 1-3 ameliorates one or more symptom or hallmark of a leukodystrophy. In certain embodiments, the leukodystrophy is AxD. In certain embodiments, the symptom or hallmark is selected from motor delays, cognitive delays, paroxysmal deterioration, seizures, vomiting, swallowing difficulties, ataxic gait, palatal myoclonus, autonomic dysfunction, and presence of intra-astrocytic inclusions called Rosenthal fibers.

In certain embodiments, an oligomeric compound complementary to any of SEQ ID NO: 1-3 is capable of reducing the detectable amount of GFAP RNA in vitro by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% when administered according to the standard cell assay. In certain embodiments, an oligomeric compound complementary to SEQ ID NO: 1, SEQ ID NO: 2915, or SEQ ID NO: 2916 is capable of decreasing the amount of GFAP in vitro by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% when administered according to the standard in vitro assay. In certain embodiments, an oligomeric compound complementary to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is capable of reducing the detectable amount of GFAP RNA in the CSF of a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, an oligomeric compound complementary to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is capable of decreasing the detectable amount of GFAP in the CSF of a subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

C. Certain Target Nucleic Acids in Certain Tissues

In certain embodiments, oligomeric compounds comprise or consist of an oligonucleotide comprising a portion that is complementary to a target nucleic acid, wherein the target nucleic acid is expressed in a pharmacologically relevant tissue. In certain embodiments, the pharmacologically relevant tissues are the cells and tissues that comprise the central nervous system (CNS). Such tissues include the brain and spinal cord. In certain embodiments, the pharmacologically relevant tissues include white matter tracts across the brain and spinal cord, such tissues include the corpus callosum, cortex, cerebellum, hippocampus, brain stem, striatum, and spinal cord. In certain embodiments, the pharmacologically relevant tissues include the cortex, cerebellum, hippocampus, brain stem, and spinal cord. In certain embodiments, the pharmacologically relevant cells are oligodendrocytes and oligodendrocyte progenitor cells. In certain embodiments, the pharmacologically relevant cells are Schwann cells or Schwann cell progenitors.

VI. Certain Pharmaceutical Compositions

In certain embodiments, described herein are pharmaceutical compositions comprising one or more oligomeric compounds. In certain embodiments, the one or more oligomeric compounds each consists of a modified oligonucleotide. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises or consists of a sterile saline solution and one or more oligomeric compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and phosphate-buffered saline (PBS). In certain embodiments, the sterile PBS is pharmaceutical grade PBS. In certain embodiments, a pharmaceutical composition comprises or consists of one or more oligomeric compound and artificial cerebrospinal fluid (“artificial CSF” or “aCSF”). In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

In certain embodiments, a pharmaceutical composition comprises a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, a pharmaceutical composition consists essentially of a modified oligonucleotide and artificial cerebrospinal fluid. In certain embodiments, the artificial cerebrospinal fluid is pharmaceutical grade.

In certain embodiments, pharmaceutical compositions comprise one or more oligomeric compound and one or more excipients. In certain embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, oligomeric compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions comprising an oligomeric compound encompass any pharmaceutically acceptable salts of the oligomeric compound, esters of the oligomeric compound, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising oligomeric compounds comprising one or more oligonucleotide, upon administration to a subject, including a human, are capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of oligomeric compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In certain embodiments, prodrugs comprise one or more conjugate group attached to an oligonucleotide, wherein the conjugate group is cleaved by endogenous nucleases within the body.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid, such as an oligomeric compound, is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions comprise one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents comprising an oligomeric compound provided herein to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, pharmaceutical compositions comprise a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, pharmaceutical compositions are prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration. In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, intrathecal (IT), intracerebroventricular (ICV), intraneural, perineural, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

Under certain conditions, certain compounds disclosed herein act as acids. Although such compounds may be drawn or described in protonated (free acid) form, or ionized and in association with a cation (salt) form, aqueous solutions of such compounds exist in equilibrium among such forms. For example, a phosphate linkage of an oligonucleotide in aqueous solution exists in equilibrium among free acid, anion and salt forms. Unless otherwise indicated, compounds described herein are intended to include all such forms. Moreover, certain oligonucleotides have several such linkages, each of which is in equilibrium. Thus, oligonucleotides in solution exist in an ensemble of forms at multiple positions all at equilibrium. The term “oligonucleotide” is intended to include all such forms. Drawn structures necessarily depict a single form. Nevertheless, unless otherwise indicated, such drawings are likewise intended to include corresponding forms. Herein, a structure depicting the free acid of a compound followed by the term “or salt thereof” expressly includes all such forms that may be fully or partially protonated/de-protonated/in association with a cation. In certain instances, one or more specific cation is identified.

In certain embodiments, modified oligonucleotides or oligomeric compounds are in aqueous solution with sodium. In certain embodiments, modified oligonucleotides or oligomeric compounds are in aqueous solution with potassium. In certain embodiments, modified oligonucleotides or oligomeric compounds are in PBS. In certain embodiments, modified oligonucleotides or oligomeric compounds are in water. In certain such embodiments, the pH of the solution is adjusted with NaOH and/or HCl to achieve a desired pH.

Herein, certain specific doses are described. A dose may be in the form of a dosage unit. For clarity, a dose (or dosage unit) of a modified oligonucleotide or an oligomeric compound in milligrams indicates the mass of the free acid form of the modified oligonucleotide or oligomeric compound. As described above, in aqueous solution, the free acid is in equilibrium with anionic and salt forms. However, for the purpose of calculating dose, it is assumed that the modified oligonucleotide or oligomeric compound exists as a solvent-free, sodium-acetate free, anhydrous, free acid. For example, where a modified oligonucleotide or an oligomeric compound is in solution comprising sodium (e.g., saline), the modified oligonucleotide or oligomeric compound may be partially or fully de-protonated and in association with Na+ ions. However, the mass of the protons are nevertheless counted toward the weight of the dose, and the mass of the Na+ ions are not counted toward the weight of the dose. Thus, for example, a dose, or dosage unit, of 10 mg of Compound No. 1362458, equals the number of fully protonated molecules that weighs 10 mg. This would be equivalent to 10.47 mg of solvent-free, sodium acetate-free, anhydrous sodiated Compound No. 1362458. When an oligomeric compound comprises a conjugate group, the mass of the conjugate group is included in calculating the dose of such oligomeric compound. If the conjugate group also has an acid, the conjugate group is likewise assumed to be fully protonated for the purpose of calculating dose.

VII. Certain Compositions

1. Compound No. 1166998

In certain embodiments, Compound No. 1166998 is characterized as a 6-10-4 MOE gapmer having a sequence (from 5′ to 3′) of CAGTATTACCTCTACTAGTC (SEQ ID NO: 20), wherein each of nucleosides 1-6 and 17-20 (from 5′ to 3′) are 2′-β-D-MOE nucleosides and each of nucleosides 7-16 are 2′-β-D-deoxynucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, and 17 to 18 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 to 2, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 18 to 19, and 19 to 20 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1166998 is represented by the following chemical notation: ^(m)C_(es)A_(eo)G_(eo)T_(eo)A_(eo)T_(eo)T_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds) ^(m)C_(ds)T_(ds)A_(ds) ^(m)C_(ds)T_(ds)A_(eo)G_(es)T_(es) ^(m)C_(es) (SEQ ID NO: 20), wherein:

-   -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1166998 is represented by the following chemical structure:

Structure 1. Compound No. 1166998

In certain embodiments, the sodium salt of Compound No. 1166998 is represented by the following chemical structure:

Structure 2. The Sodium Salt of Compound No. 1166998

2. Compound No. 1166985

In certain embodiments, Compound No. 1166985 is characterized as a 6-10-4 MOE gapmer having a sequence (from 5′ to 3′) of CACATTCACTAATATTTAAC (SEQ ID NO: 21), wherein each of nucleosides 1-6 and 17-20 (from 5′ to 3′) are 2′-β-D-MOE nucleosides and each of nucleosides 7-16 are 2′-β-D-deoxynucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, and 17 to 18 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 to 2, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 18 to 19, and 19 to 20 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1166985 is represented by the following chemical notation: ^(m)C_(es)A_(eo) ^(m)C_(eo)A_(eo)T_(eo)T_(eo) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)A_(ds)A_(ds)T_(ds)A_(ds)T_(ds)T_(ds)T_(eo)A_(es)A_(es) ^(m)C_(es) (SEQ ID NO: 21), wherein:

-   -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1166985 is represented by the following chemical structure:

Structure 3. Compound No. 1166985

In certain embodiments, the sodium salt of Compound No. 1166985 is represented by the following chemical structure:

Structure 4. The Sodium Salt of Compound No. 1166985

3. Compound No. 1166954

In certain embodiments, Compound No. 1166954 is characterized as a 5-10-5 MOE gapmer having a sequence (from 5′ to 3′) of CCAGTGTCTTCACTTTGCTC (SEQ ID NO: 825), wherein each of nucleosides 1-5 and 16-20 (from 5′ to 3′) are 2′-β-D-MOE nucleosides and each of nucleosides 6-15 are 2′-β-D-deoxynucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 16 to 17, and 17 to 18 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 to 2, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 18 to 19, and 19 to 20 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1166954 is represented by the following chemical notation: ^(m)C_(es) ^(m)C_(eo)A_(eo)G_(eo)T_(eo)G_(ds)T_(ds) ^(m)C_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(eo)G_(eo) ^(m)C_(es)T_(es) ^(m)C_(e) (SEQ ID NO: 825), wherein:

-   -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1166954 is represented by the following chemical structure:

Structure 5: Compound No. 1166954

In certain embodiments, the sodium salt of Compound No. 1166954 is represented by the following chemical structure:

Structure 6: The Sodium Salt of Compound No. 1166954

4. Compound No. 1072813

In certain embodiments, Compound No. 1072813 is characterized as a 5-10-5 MOE gapmer having a sequence (from 5′ to 3′) of GCAACAGTTTCCATAACAAC (SEQ ID NO: 1499), wherein each of nucleosides 1-5 and 16-20 (from 5′ to 3′) are 2′-β-D-MOE nucleosides and each of nucleosides 6-15 are 2′-β-D-deoxynucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 16 to 17, and 17 to 18 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 18 to 19, and 19 to 20 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1072813 is represented by the following chemical notation: G_(es) ^(m)C_(eo)A_(eo)A_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)T_(ds)A_(ds)A_(eo) ^(m)C_(eo)A_(es)A_(es) ^(m)Ce (SEQ ID NO: 1499), wherein:

-   -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1072813 is represented by the following chemical structure:

Structure 7: Compound No. 1072813

In certain embodiments, the sodium salt of Compound No. 1072813 is represented by the following chemical structure:

Structure 8: The Sodium Salt of Compound No. 1072813

5. Compound No. 1199983

In certain embodiments, Compound No. 1199983 is characterized as a 5-10-5 MOE gapmer having a sequence (from 5′ to 3′) of TGGTCCTAAATATTCTAGTC (SEQ ID NO: 2170), wherein each of nucleosides 1-5 and 16-20 (from 5′ to 3′) are 2′-β-D-MOE nucleosides and each of nucleosides 6-15 are 2′-β-D-deoxynucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 5 to 6, 16 to 17, and 17 to 18 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 to 2, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 18 to 19, and 19 to 20 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1199983 is represented by the following chemical notation: T_(es)G_(eo)G_(eo)T_(eo) ^(m)C_(eo) ^(m)C_(ds)T_(ds)A_(ds)A_(ds)A_(ds)T_(ds)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)T_(eo)A_(eo)G_(es)T_(es) ^(m)C_(e) (SEQ ID NO: 2170), wherein:

-   -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1199983 is represented by the following chemical structure:

Structure 9: Compound No. 1199983

In certain embodiments, the sodium salt of Compound No. 1199983 is represented by the following chemical structure:

Structure 10: The Sodium Salt of Compound No. 1199983

6. Compound No. 1166721

In certain embodiments, Compound No. 1166721 is characterized as a 5-8-5 MOE gapmer having a sequence (from 5′ to 3′) of TGGTCCTAAATATTCTAGTC (SEQ ID NO: 2818), wherein each of nucleosides 1-5 and 14-18 (from 5′ to 3′) are 2′-β-D-MOE nucleosides and each of nucleosides 6-15 are 2′-β-D-deoxynucleosides, wherein the internucleoside linkages between nucleosides 2 to 3, 3 to 4, 4 to 5, 14 to 15 and 15 to 16 are phosphodiester internucleoside linkages and the internucleoside linkages between nucleosides 1 to 2, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 16 to 17, and 17 to 18 are phosphorothioate internucleoside linkages, and wherein each cytosine is a 5-methyl cytosine.

In certain embodiments, Compound No. 1166721 is represented by the following chemical notation: T_(es)G_(eo)G_(eo) ^(m)C_(eo)A_(es)G_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(eo) ^(m)C_(eo)T_(es)A_(es) ^(m)C_(e) (SEQ ID NO: 2818), wherein:

-   -   A=an adenine nucleobase,     -   ^(m)C=a 5-methyl cytosine nucleobase,     -   G=a guanine nucleobase,     -   T=a thymine nucleobase,     -   e=a 2′-β-D-MOE sugar moiety,     -   d=a 2′-β-D-deoxyribosyl sugar moiety,     -   s=a phosphorothioate internucleoside linkage, and     -   o=a phosphodiester internucleoside linkage.

In certain embodiments, Compound No. 1166721 is represented by the following chemical structure:

Structure 11: Compound No. 1166721

In certain embodiments, the sodium salt of Compound No. 1166721 is represented by the following chemical structure:

Structure 12: The Sodium Salt of Compound No. 1166721

VIII. Certain Hotspot Regions

In certain embodiments, nucleobases in the ranges specified below comprise a hotspot region of GFAP nucleic acid. In certain embodiments, modified oligonucleotides that are complementary to a hotspot region of GFAP nucleic acid achieve an average of more than 50% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides that are complementary to a hotspot region of GFAP nucleic acid achieve an average of 75% or greater reduction of GFAP RNA in vivo in the standard in vivo assay.

1. Nucleobases 9324-9348 of SEQ ID NO: 2

In certain embodiments, nucleobases 9324-9348 of SEQ ID NO: 2 comprise a hotspot region. In certain embodiments, modified oligonucleotides are complementary to a portion of nucleobases 9324-9348 of SEQ ID NO: 2.

In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are 18 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmers are MOE gapmers. In certain embodiments, the internucleoside linkages of the modified oligonucleotides are phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in order from 5′ to 3′: In certain embodiments, modified nucleotides have an internucleoside linkage motif of sooosssssssssssooss, sooooossssssssssoss, soooossssssssssooss, sooosssssssssooss, or sooossssssssssoooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage.

The nucleobase sequences of SEQ ID Nos: 21, 1177, 2321, 2398, 2808-2809, 2840-2842, and 2853-2854 are complementary to a portion of nucleobases 9324-9348 of SEQ ID NO: 2.

The nucleobase sequence of Compound Nos.: 1048181-1048182, 1104071-1104072, 1166746-1166748, 1166803-1166808, 1166894-1166899, 1166985-1166990, 1174016, and 1174018 are complementary to a portion of nucleobases 9324-9348 of SEQ ID NO: 2.

In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9324-9348 of SEQ ID NO: 2 achieve at least 7% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9324-9348 of SEQ ID NO: 2 achieve an average of 42% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9324-9348 of SEQ ID NO: 2 achieve an average of 81% reduction of GFAP RNA in vivo in the standard in vivo assay.

2. Nucleobases 9459-9480 of SEQ ID NO: 2

In certain embodiments, nucleobases 9459-9480 of SEQ ID NO: 2 comprise a hotspot region. In certain embodiments, modified oligonucleotides are complementary to a portion of nucleobases 9459-9480 of SEQ ID NO: 2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are 18 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmers are MOE gapmers. In certain embodiments, the internucleoside linkages of the modified oligonucleotides are phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in order from 5′ to 3′: In certain embodiments, modified nucleotides have an internucleoside linkage motif of sooosssssssssssooss or soooossssssssssooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage.

The nucleobase sequences of SEQ ID Nos: 555, 2093, 2170, and 2813 are complementary to a portion of nucleobases 9459-9480 of SEQ ID NO: 2.

The nucleobase sequence of Compound Nos.: 1048190, 1104116-1104117, and 1199982-1199984 are complementary to a portion of nucleobases 9459-9480 of SEQ ID NO: 2.

In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9459-9480 of SEQ ID NO: 2 achieve at least 26% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9459-9480 of SEQ ID NO: 2 achieve an average of 42% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9459-9480 of SEQ ID NO: 2 achieve an average of 91% reduction of GFAP RNA in vivo in the standard in vivo assay.

3. Nucleobases 9530-9580 of SEQ ID NO: 2

In certain embodiments, nucleobases 9530-9580 of SEQ ID NO: 2 comprise a hotspot region. In certain embodiments, modified oligonucleotides are complementary to a portion of nucleobases 9530-9580 of SEQ ID NO: 2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are 18 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmers are MOE gapmers. In certain embodiments, the internucleoside linkages of the modified oligonucleotides are phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in order from 5′ to 3′: In certain embodiments, modified nucleotides have an internucleoside linkage motif of sooosssssssssssooss, sooooossssssssssoss, soooossssssssssooss, sooosssssssssooss, or sooossssssssssoooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage.

The nucleobase sequences of SEQ ID Nos.: 20, 88, 166, 1331, 1408, 1485, 1637, 1713, 1714, 1789, 1790, 1637, 1638, 1865, 1866, 1941, 2018, 2095, 2172, 2249, 2326, 2403, 2480, 2557, 2633, 2709, 2785, 2816-2818, 2859, 2861, and 2886-2887 are complementary to a portion of nucleobases 9530-9580 of SEQ ID NO: 2.

The nucleobase sequence of Compound Nos.: 1048200-1048201, 1073062-1073064, 1104142-1104161, 1166719-1166721, 1166816-1166823, 1166826, 1166907-1166920, 1166998-1167011, 1174024, 1174026, and 1174029-1174030 are complementary to a portion of nucleobases 9530-9580 of SEQ ID NO: 2.

In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9530-9580 of SEQ ID NO: 2 achieve at least 27% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9530-9580 of SEQ ID NO: 2 achieve an average of 52% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 9530-9580 of SEQ ID NO: 2 achieve an average of 82% reduction of GFAP RNA in vivo in the standard in vivo assay.

4. Nucleobases 12006-12038 of SEQ ID NO: 2

In certain embodiments, nucleobases 12006-12038 of SEQ ID NO: 2 comprise a hotspot region. In certain embodiments, modified oligonucleotides are complementary to a portion of nucleobases 12006-12038 of SEQ ID NO: 2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are 18 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmers are MOE gapmers. In certain embodiments, the internucleoside linkages of the modified oligonucleotides are phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in order from 5′ to 3′: In certain embodiments, modified nucleotides have an internucleoside linkage motif of sooosssssssssssooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage.

The nucleobase sequences of SEQ ID Nos.: 815, 893, 971, 1049, 1269, 1270, 1346, 1423, 1499, 1500, 1660, 1736, 2655, and 2731 are complementary to a portion of nucleobases 12006-12038 of SEQ ID NO: 2.

The nucleobase sequence of Compound Nos.: 1047362-1047365, 1072813-1072818, and 1103276-1103279 are complementary to a portion of nucleobases 12006-12038 of SEQ ID NO: 2.

In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 12006-12038 of SEQ ID NO: 2 achieve at least 29% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 12006-12038 of SEQ ID NO: 2 achieve an average of 52% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 12006-12038 of SEQ ID NO: 2 achieve an average of 82% reduction of GFAP RNA in vivo in the standard in vivo assay.

5. Nucleobases 13038-13058 of SEQ ID NO: 2

In certain embodiments, nucleobases 13038-13058 of SEQ ID NO: 2 comprise a hotspot region. In certain embodiments, modified oligonucleotides are complementary to a portion of nucleobases 13038-13058 of SEQ ID NO: 2. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are 18 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, the gapmers are MOE gapmers. In certain embodiments, the internucleoside linkages of the modified oligonucleotides are phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In certain embodiments, the phosphodiester (“o”) and phosphorothioate (“s”) internucleoside linkages are arranged in order from 5′ to 3′: In certain embodiments, modified nucleotides have an internucleoside linkage motif of sooosssssssssssooss, sooooossssssssssoss, or soooossssssssssooss, wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage.

The nucleobase sequences of SEQ ID Nos.: 825 and 1973 are complementary to a portion of nucleobases 13038-13058 of SEQ ID NO: 2.

The nucleobase sequence of Compound Nos.: 1047522, 1166954, and 1167046 are complementary to a portion of nucleobases 13038-13058 of SEQ ID NO: 2.

In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 13038-13058 of SEQ ID NO: 2 achieve at least 27% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 13038-13058 of SEQ ID NO: 2 achieve an average of 41% reduction of GFAP RNA in vitro in the standard cell assay. In certain embodiments, modified oligonucleotides complementary to a portion of nucleobases 13038-13058 of SEQ ID NO: 2 achieve an average of 84% reduction of GFAP RNA in vivo in the standard in vivo assay.

6. Additional Hotspot Regions

In certain embodiments, the ranges described in the Table below comprise hotspot regions. Each hotspot region begins with the nucleobase of SEQ ID NO: 2 identified in the “Start Site SEQ ID NO: 2” column and ends with the nucleobase of SEQ ID NO: 2 identified in the “Stop Site SEQ ID NO: 2” column. In certain embodiments, modified oligonucleotides are complementary within any of the hotspot regions 1-14, as defined in the table below. In certain embodiments, modified oligonucleotides are 18 nucleobases in length. In certain embodiments, modified oligonucleotides are 20 nucleobases in length. In certain embodiments, modified oligonucleotides are gapmers. In certain embodiments, modified oligonucleotides are 5-8-5, 5-10-3, 4-10-6, 6-10-4, or 5-10-5 MOE gapmers.

The nucleobase sequence of compounds listed in the “Compound No. in range” column in the table below are complementary to SEQ ID NO: 2 within the specified hotspot region. The nucleobase sequence of the oligonucleotides listed in the “SEQ ID NO: in range” column in the table below are complementary to the target sequence, SEQ ID NO: 2, within the specified hotspot region.

In certain embodiments, modified oligonucleotides complementary to nucleobases within the hotspot region achieve at least “Min. % Red. in vitro” (minimum % reduction, relative to untreated control cells) of GFAP RNA in vitro in the standard cell assay, as indicated in the table below. In certain embodiments, modified oligonucleotides complementary to nucleobases within the hotspot region achieve an average of “Avg. % Red. in vitro” (average % reduction, relative to untreated control cells) of GFAP RNA in vitro in the standard cell assay, as indicated in the table below. In certain embodiments, modified oligonucleotides complementary to nucleobases within the hotspot region achieve a maximum of “Max. % Red. in vitro” (maximum % reduction, relative to untreated control cells) of GFAP RNA in vitro in the standard cell assay, as indicated in the table below. In certain embodiments, modified oligonucleotides complementary to nucleobases within the hotspot region achieve an average of “Avg. % Red. in vivo” (average % reduction, relative to PBS-treated animals) of GFAP RNA in vivo in the standard in vivo assay, as indicated in the table below.

TABLE 1 GFAP Hotspots Start Start Site Site Min. Max. Avg. Avg. Hotspot SEQ ID SEQ ID % Red. % Red. % Red. % Red. Compound No. SEQ ID NOs: ID NO: 2 NO: 2 in vitro In vitro in vitro in vivo in Range in Range 1 9324 9348 7 78 42 81 1048181-1048182, 21, 1177, 2321, 2398, 1104071-1104072, 2808-2809, 2840-2842, 1166746-1166748, 2853-2854 1166803-1166808, 1166894-1166899, 1166985-1166990, 1174016, 1174018 2 9459 9480 26 65 42 91 1048190, 555, 2093, 2170, 1104116-1104117, 2813 1199982-1199984 3 9530 9580 18 82 52 82 1048199-1048201, 20, 88, 166, 1331, 1073062-1073064, 1408, 1485, 1637, 1104142-1104161, 1713, 1714, 1789, 1166719-1166721, 1790, 1637, 1638, 1166816-1166823, 1865, 1866, 1941, 1166826, 2018, 2095, 2172, 1166907-1166920, 2249, 2326, 2403, 1166998-1167011, 2480, 2557, 2633, 1174024, 1174026, 2709, 2785, 1174029-1174030 2816-2818, 2859, 2861, 2886-2887 4 12006 12038 29 75 52 82 1047362-1047365, 815, 893, 971, 1049, 1072813-1072818, 1269, 1270, 1346, 1103276-1103279 1423, 1499, 1500, 1660, 1736, 2655, 2731 5 13038 13058 27 56 41 84 1047522, 1166954, 825, 1973 1167046 6 8530 8557 14 79 52 89 1047706-1047708, 213, 291, 369, 1601, 1103567-1103571 1753, 1829, 1905, 1982 7 8731 8754 44 64 53 88 1047733-1047735, 1072, 1149, 1227, 1103591, 1103592, 2291, 2368 1174050, 1174051, 1174056, 1174058, 1174062,1174063 8 8749 8807 25 92 52 75 1047601-1047610, 51, 129, 207, 752, 1072854-1072868, 830, 908, 986, 1064, 1103462-1103472, 1141, 1219, 1279, 1166738-1166740, 1280-1282, 1356-1359, 1166742, 1166744, 1433-1436, 1510-1512, 1166793-1166795, 1595, 1671, 1747, 1166798-1166800, 2206, 2283, 2360, 1166885-1166890, 2437, 2514, 2590, 1166975, 1166982, 2666, 2742, 2835-2837, 1174012-1174013 2839, 2850-2851, 2866 9 9511 9536 32 90 55 86 1048197, 1073060, 1100, 1484, 1864, 1104129-1104133, 1940, 2017, 2094, 1166749-1166751, 2171, 2819-2821, 1166809-1166810, 2856 1166812-1166813, 1166900-1166903, 1166991-1166994, 1174020 10 9565 9602 20 75 53 82 1048202-1048204, 244, 322, 400, 1562, 1073065, 1639, 1715, 1791, 1104165-1104179, 1867, 1943, 2020, 1166757-1166760, 2173, 2250, 2327, 1166831, 1166835, 2404, 2481, 2558, 1166926-1166929, 2634, 2710, 2786, 1167012, 1167017, 2873-2876, 2888, 1167018, 1174031, 2891 1174034 11 11155 11184 37 82 69 85 1073093-1073095, 1339, 1569, 2028, 1104307-1104312, 2105, 2182, 2259, 1167024-1167027, 2336, 2413, 1166761-1166763, 2822-2824, 2863 1166842-1166843, 1166845-1166847, 1166933-1166934, 1167024-1167027, 1174037 12 12044 12067 49 67 58 85 1047372-1047374, 348, 426, 504, 1425, 1072824-1072825, 1502 1166852, 1167033-1167036 13 12080 12108 45 88 68 79 1047384-1047388, 37, 115, 193, 271, 1072834-1072835, 349, 1274, 1351, 1103284-1103285, 2041, 2118 1166948, 1167040 14 13333 13367 53 93 75 89 1047579-104592, 50, 283, 361, 439, 1072849-1072850, 517, 595, 673, 751, 1166775-1166787, 829, 907, 985, 1063, 1166867, 1140, 1218, 1278, 1166869-1166875, 1508, 2825-2833, 1166877-1166878, 2843-2846 1167050-1167051, 1167053-1167060

Nonlimiting Disclosure and Incorporation by Reference

Each of the literature and patent publications listed herein is incorporated by reference in its entirety.

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar moiety (2′-OH in place of one 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of a uracil of RNA). Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified nucleobases, such as “ATmCGAUCG,” wherein ^(m)C indicates a cytosine base comprising a methyl group at the 5-position.

Certain compounds described herein (e.g., modified oligonucleotides) have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), as a or such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms, unless specified otherwise. Likewise, all cis- and trans-isomers and tautomeric forms of the compounds herein are also included unless otherwise indicated. Oligomeric compounds described herein include chirally pure or enriched mixtures as well as racemic mixtures. For example, oligomeric compounds having a plurality of phosphorothioate internucleoside linkages include such compounds in which chirality of the phosphorothioate internucleoside linkages is controlled or is random. Unless otherwise indicated, compounds described herein are intended to include corresponding salt forms.

The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the ¹H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include but are not limited to: ²H or ³H in place of 41, ¹³C or ¹⁴C in place of ¹²C, ¹⁵N in place of ¹⁴N, ¹⁷O or ¹⁸O in place of ¹⁶O and ³³S, ³⁴S, ³⁵S, or ³⁶S in place of ³²S. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.

EXAMPLES

The following examples illustrate certain embodiments of the present disclosure and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments.

Example 1: Effect of 5-10-5 MOE Gapmer Modified Oligonucleotides on Human GFAP RNA In Vitro, Single Dose

Modified oligonucleotides complementary to human GFAP nucleic acid were designed and tested for their single dose effects on GFAP RNA in vitro. The modified oligonucleotides were tested in a series of experiments that had similar culture conditions.

The modified oligonucleotides in the tables below are 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages. The gapmers are 20 nucleosides in length, wherein the central gap segment consists of ten 2′-β-D-deoxynucleosides and the 5′ and 3′ wing segments each consists of five 2′-MOE modified nucleosides. The sugar motif for the gapmers is (from 5′ to 3′): eeeeeddddddddddeeeee; wherein ‘d’ represents a 2′-β-D-deoxyribosyl sugar moiety, and ‘e’ represents a 2′-MOE sugar moiety. The internucleoside linkage motif for the gapmers is (from 5′ to 3′): sooosssssssssssooss; wherein each ‘o’ represents a phosphodiester internucleoside linkage and each ‘s’ represents a phosphorothioate internucleoside linkage. Each cytosine residue is a 5-methyl cytosine.

“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. Each modified oligonucleotide listed in the Tables below is 100% complementary to SEQ ID NO: 1 (GENBANK Accession No. NM_002055.4), or SEQ ID NO: 2 (GENBANK Accession No. NC_000017.11 truncated from nucleotides 44903001 to 44919000). ‘N/A’ indicates that the modified oligonucleotide is not 100% complementary to that particular target nucleic acid sequence.

Cultured U251 cells were treated with modified oligonucleotide at a concentration of 4,000 nM using free uptake at a density of 10,000 cells per well. After a treatment period of approximately 48 hours, total RNA was isolated from the cells and GFAP RNA levels were measured by quantitative real-time RTPCR. GFAP RNA levels were measured by human GFAP primer probe set RTS37485 (forward sequence CTGGAGGTTGAGAGGGACA, designated herein as SEQ ID NO: 11; reverse sequence GCTTCATCTGCTTCCTGTCT, designated herein as SEQ ID NO: 12; probe sequence CTGGAGCTTCTGCCTCACAGTGG, designated herein as SEQ ID NO: 13). GFAP RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Reduction of GFAP RNA is presented in the tables below as percent GFAP RNA amount relative to untreated control cells. Each table represents results from an individual assay plate. The values marked with an asterisk (*) indicate that the modified oligonucleotide is complementary to the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of the modified oligonucleotides complementary to the amplicon region.

TABLE 2 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 GFAP SEQ Compound Start Stop Start Stop RNA ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047144   17   36  3465  3484 CGAGGGCTTTATGAAGGAGT  80 22 1047160  131  150  3579  3598 GCCAGGAGCCAGGCCCCCCA  67 23 1047176  248  267  3696  3715 GGCCCGGGTCTCCTTGAAGC 109 24 1047192  488  507  3936  3955 GTCCTGTGCCAGATTGTCCC   7* 25 1047208  579  598 N/A N/A CATCTGCTTCCTGTCTATAG   6* 26 1047224  649  668  5238  5257 CTCAAGAACCGGATCTCCTC 105 27 1047240  813  832  5699  5718 ACTCTTCGGCTTCATGCATG  69 28 1047256 1029 1048  7601  7620 GCGCCTCCTGATAACTGGCC  80 29 1047272 1251 1270 10865 10884 CTTCTGACACAGACTTGGTG  92 30 1047288 1298 1317 10912 10931 ATCCCGCATCTCCACGGTCT  90 31 1047304 1351 1370 11640 11659 CTGCCTCACATCACATCCTT  87 32 1047320 1416 1435 11705 11724 CGGAGCAACTATCCTGCTTC 102 33 1047336 1617 1636 11906 11925 AAGCTGCTGGCCATGCCCCT  87 34 1047352 1664 1683 11953 11972 TGCCCCCCGCCCTCCTCCCC  83 35 1047368 1736 1755 12025 12044 GAGAGAACCTCCATCTCTGG  47 36 1047384 1791 1810 12080 12099 TCAGTTTTCCTCCAGCAGCC  55 37 1047400 1854 1873 12143 12162 GACAAAACAAGCCTCTGGCC  66 38 1047416 2036 2055 12325 12344 CGTCCCCACCCATCTTAGAC  60 39 1047432 2180 2199 12469 12488 GTGCTGAGAATCAAGCTCCC  93 40 1047448 2237 2256 12526 12545 CCCCCTCTATCCCTCCCAGC  11 41 1047464 2280 2299 12569 12588 CTGGGCTTGACCTCTCTGTA  63 42 1047480 2379 2398 12668 12687 TGGTCACCCACAACCCCTAC  81 43 1047496 2457 2476 12746 12765 CCCTTTCTCTCCTGTTTCAG  79 44 1047512 2486 2505 12775 12794 AAGTCATGCCCTGCCCCCAT  45 45 1047528 2780 2799 13069 13088 GCACCCGGCCTCCAGGCTGC  76 46 1047544 2861 2880 13150 13169 GGCACAGATCCCACCAGTCT 106 47 1047560 2903 2922 13192 13211 GAGAGGAGAACCCTGAAGTG  72 48 1047576 3035 3054 13324 13343 CCTCAGCGACTAAAGGCAGC  77 49 1047592 3058 3077 13347 13366 GCGCAGCATTTGTCTTTATT  47 50 1047608 N/A N/A  8777  8796 GCTTTTGAGATATCTTGTGA   8 51 1047624 N/A N/A  9031  9050 GTTTAATGTACAGTTACTCT  71 52 1047640 N/A N/A  9070  9089 CCAAGGACTCACCACCTTTA  75 53 1047656 N/A N/A  9202  9221 AGGGATGAAAGAATAAAGCA  92 54 1047672 N/A N/A  8381  8400 CCTGCTGTACTGACCTCGAA  94 55 1047688 N/A N/A  8456  8475 ATCCTCAGTCCCAGTCTGGA  54 56 1047704 N/A N/A  8504  8523 CTGCAGTGTCACGAAGGCCC  74 57 1047720 N/A N/A  8637  8656 TGTCAAGCTCTCACCCAGTT  77 58 1047736 N/A N/A  8737  8756 TTGGTGCTTTTGCCCCCTGT  56 59 1047752 N/A N/A  4093  4112 GGATAGTGCCCCATCAAGAG 109 60 1047768 N/A N/A  4264  4283 AGTCACAAAGCCCAGCCATG  95 61 1047784 N/A N/A  4322  4341 GCTTCCAACTCCTCCTTTAT 101 62 1047800 N/A N/A  4359  4378 CAGAATCCAATCTCCCTCAT 106 63 1047816 N/A N/A  4405  4424 GCTTTGCGCCCAGACCTGCC  68 64 1047832 N/A N/A  4525  4544 ATTCCTCTGATCCCAGGTAA  52 65 1047848 N/A N/A  4704  4723 CCTTAACTCATTACTAAGGT  69 66 1047864 N/A N/A  4806  4825 GAGACCACCCCCACCCAGGA  70 67 1047880 N/A N/A  4868  4887 GTCCAGGCTCTTCTGAGGAC  66 68 1047896 N/A N/A  5007  5026 GTGGCCATCAATCCTTTCCT 103 69 1047912 N/A N/A  5117  5136 CCCCAGGCTCCTTCTCCCCA  74 70 1047928 N/A N/A  5388  5407 TGTCTCTACCTGCCAATCTC 100 71 1047944 N/A N/A  5521  5540 CTCAGGGTACAGGCCACAGC  90 72 1047960 N/A N/A  5791  5810 ACCCTCCTTCCCCCATTCTC  94 73 1047976 N/A N/A  5934  5953 AGCTACTACTAATAATAGCA  98 74 1047992 N/A N/A  6023  6042 ACTTCGGCTCTCTCATCTGT  75 75 1048008 N/A N/A  6146  6165 AACCCAAAACAGACTGGCAG  79 76 1048024 N/A N/A  6281  6300 CCCACACTACATATAAGCTC 105 77 1048040 N/A N/A  6329  6348 CCTGTCCTGCCTAGCCCAAA  71 78 1048056 N/A N/A  6417  6436 GGGCCCTGCCTCTCTGTGCT  81 79 1048072 N/A N/A  6544  6563 ATAGCCCTTTCTCCCCTGCC  86 80 1048088 N/A N/A  6959  6978 AATCCAGAACCTTCCACACT 114 81 1048104 N/A N/A  7073  7092 TGGGACTTTTCCCAACAACT  93 82 1048120 N/A N/A  7412  7431 CGCCCTCGACCCAGGTCCTC  57 83 1048136 N/A N/A  7907  7926 AGTGACTGCCTGCTATGTGT  94 84 1048152 N/A N/A  7989  8008 TTGGAGGGTGACCCAAGTCC  81 85 1048168 N/A N/A  8235  8254 ACGCCCTTTTCCTTGCCAGG  74 86 1048184 N/A N/A  9373  9392 TAGCTCCCCCCTCCCCCCGC  52 87 1048200 N/A N/A  9534  9553 GCAGTATTACCTCTACTAGT  45 88 1048216 N/A N/A  9610  9629 CCTGTCCCCTTTCCTCTTTC  74 89 1048232 N/A N/A  9791  9810 CCACCAACCAGCCACATGAC  98 90 1048248 N/A N/A  9826  9845 ATCAGGAGACCAGAGCTCAA  78 91 1048264 N/A N/A 10622 10641 GGCCTGGCTTCATTTCAGCC  66 92 1048280 N/A N/A 10734 10753 AGTATGAGAACCTATGCAAC  80 93 1048296 N/A N/A 10793 10812 GGGCATGAGCCATCCTCTCC  35 94 1048312 N/A N/A 10942 10961 GACTGGGCCCAAATCCCTCC  87 95 1048328 N/A N/A 11063 11082 CCTTGCTCTCCTCCAGAATT  67 96 1048344 N/A N/A 11227 11246 GCTACTAACTTTAATTCTCT  41 97 1048360 N/A N/A 11321 11340 CCTCTTCCCATTCCCCTGGT  62 98 1048376 N/A N/A 11488 11507 GGTCTTACTTTTCTTGATAG  72 99

TABLE 3 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 GFAP SEQ Compound Start Stop Start Stop RNA ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047145   18   37  3466  3485 GCGAGGGCTTTATGAAGGAG  99 100 1047161  133  152  3581  3600 CGGCCAGGAGCCAGGCCCCC  82 101 1047177  267  286  3715  3734 TCATCTCTGCCCGCTCACTG  88 102 1047193  490  509  3938  3957 AGGTCCTGTGCCAGATTGTC   8* 103 1047209  585  604 N/A N/A TGGCTTCATCTGCTTCCTGT   5* 104 1047225  651  670  5240  5259 TCCTCAAGAACCGGATCTCC  37 105 1047241  821  840  5707  5726 GCGGTACCACTCTTCGGCTT  77 106 1047257 1051 1070  7623  7642 CCCTCTTCCTCCAGCCGCGC  35 107 1047273 1252 1271 10866 10885 CCTTCTGACACAGACTTGGT  86 108 1047289 1305 1324 10919 10938 CCTCTCCATCCCGCATCTCC  87 109 1047305 1352 1371 11641 11660 CCTGCCTCACATCACATCCT  66 110 1047321 1417 1436 11706 11725 GCGGAGCAACTATCCTGCTT  96 111 1047337 1631 1650 11920 11939 CCTCATTCTAACGCAAGCTG  58 112 1047353 1665 1684 11954 11973 GTGCCCCCCGCCCTCCTCCC  46 113 1047369 1739 1758 12028 12047 TCCGAGAGAACCTCCATCTC  72 114 1047385 1792 1811 12081 12100 CTCAGTTTTCCTCCAGCAGC  50 115 1047401 1876 1895 12165 12184 TCCCACCTCATAAAAACCAA  94 116 1047417 2059 2078 12348 12367 GGTGACTGCCCCAGGTGGCA  68 117 1047433 2181 2200 12470 12489 AGTGCTGAGAATCAAGCTCC  75 118 1047449 2238 2257 12527 12546 CCCCCCTCTATCCCTCCCAG  60 119 1047465 2310 2329 12599 12618 GTCCCCTCCAGTCTGCACGG  49 120 1047481 2380 2399 12669 12688 CTGGTCACCCACAACCCCTA  82 121 1047497 2458 2477 12747 12766 CCCCTTTCTCTCCTGTTTCA  29 122 1047513 2491 2510 12780 12799 GGACAAAGTCATGCCCTGCC  85 123 1047529 2781 2800 13070 13089 AGCACCCGGCCTCCAGGCTG  86 124 1047545 2862 2881 13151 13170 GGGCACAGATCCCACCAGTC  65 125 1047561 2924 2943 13213 13232 CCCTTCTTCGGCCTTAGAGG  62 126 1047577 3040 3059 13329 13348 TTTTTCCTCAGCGACTAAAG  49 127 1047593 3060 3079 13349 13368 GGGCGCAGCATTTGTCTTTA  59 128 1047609 N/A N/A  8778  8797 GGCTTTTGAGATATCTTGTG  13 129 1047625 N/A N/A  9036  9055 TGCCAGTTTAATGTACAGTT  72 130 1047641 N/A N/A  9072  9091 ACCCAAGGACTCACCACCTT  89 131 1047657 N/A N/A  9212  9231 ATGGAGCCTCAGGGATGAAA  69 132 1047673 N/A N/A  8382  8401 CCCTGCTGTACTGACCTCGA  91 133 1047689 N/A N/A  8460  8479 CCTGATCCTCAGTCCCAGTC  82 134 1047705 N/A N/A  8506  8525 CGCTGCAGTGTCACGAAGGC  63 135 1047721 N/A N/A  8643  8662 GGCAGATGTCAAGCTCTCAC  83 136 1047737 N/A N/A  3963  3982 TCCTCACTTCTGCCTCACAG  66 137 1047753 N/A N/A  4095  4114 AAGGATAGTGCCCCATCAAG  69 138 1047769 N/A N/A  4265  4284 CAGTCACAAAGCCCAGCCAT  64 139 1047785 N/A N/A  4324  4343 CCGCTTCCAACTCCTCCTTT  49 140 1047801 N/A N/A  4361  4380 CCCAGAATCCAATCTCCCTC  85 141 1047817 N/A N/A  4419  4438 AGGCAGTCACCTGTGCTTTG  96 142 1047833 N/A N/A  4526  4545 GATTCCTCTGATCCCAGGTA  63 143 1047849 N/A N/A  4705  4724 GCCTTAACTCATTACTAAGG  82 144 1047865 N/A N/A  4807  4826 AGAGACCACCCCCACCCAGG  91 145 1047881 N/A N/A  4910  4929 TGGTTTCATCCTGGAGCCTG  60 146 1047897 N/A N/A  5012  5031 GGTGGGTGGCCATCAATCCT  71 147 1047913 N/A N/A  5168  5187 CATCTGCTTCCTGGAGTGGC  31 148 1047929 N/A N/A  5400  5419 TCTTTCATTTCCTGTCTCTA  81 149 1047945 N/A N/A  5561  5580 CCCGAACCTCCTGACCAGGG 117 150 1047961 N/A N/A  5800  5819 GCTTCCTCCACCCTCCTTCC  75 151 1047977 N/A N/A  5935  5954 CAGCTACTACTAATAATAGC 104 152 1047993 N/A N/A  6040  6059 TTAGTTAACCTCTCTGGACT  79 153 1048009 N/A N/A  6147  6166 TAACCCAAAACAGACTGGCA 101 154 1048025 N/A N/A  6282  6301 TCCCACACTACATATAAGCT  83 155 1048041 N/A N/A  6330  6349 GCCTGTCCTGCCTAGCCCAA  80 156 1048057 N/A N/A  6464  6483 GCCACTCACACTCCTCAGCT  79 157 1048073 N/A N/A  6548  6567 TCTAATAGCCCTTTCTCCCC  88 158 1048089 N/A N/A  6961  6980 AGAATCCAGAACCTTCCACA  88 159 1048105 N/A N/A  7074  7093 CTGGGACTTTTCCCAACAAC  86 160 1048121 N/A N/A  7418  7437 AGGCCCCGCCCTCGACCCAG  95 161 1048137 N/A N/A  7915  7934 AAACATCTAGTGACTGCCTG  87 162 1048153 N/A N/A  8061  8080 AAGGAGGCAGAAGAGATGGG  59 163 1048169 N/A N/A  8286  8305 CTGGGACACCCCTAGGCTGG  68 164 1048185 N/A N/A  9374  9393 TTAGCTCCCCCCTCCCCCCG  84 165 1048201 N/A N/A  9536  9555 TGGCAGTATTACCTCTACTA  43 166 1048217 N/A N/A  9611  9630 TCCTGTCCCCTTTCCTCTTT  63 167 1048233 N/A N/A  9792  9811 GCCACCAACCAGCCACATGA  89 168 1048249 N/A N/A 10005 10024 CTGTAATCCCCTTACTCGGG  94 169 1048265 N/A N/A 10636 10655 CTGCTCTGTCTTCTGGCCTG  65 170 1048281 N/A N/A 10735 10754 GAGTATGAGAACCTATGCAA  53 171 1048297 N/A N/A 10794 10813 AGGGCATGAGCCATCCTCTC  78 172 1048313 N/A N/A 10944 10963 CTGACTGGGCCCAAATCCCT  95 173 1048329 N/A N/A 11074 11093 ACATTCAGTTTCCTTGCTCT  89 174 1048345 N/A N/A 11228 11247 AGCTACTAACTTTAATTCTC  82 175 1048361 N/A N/A 11330 11349 GCCAAATCCCCTCTTCCCAT  19 176 1048377 N/A N/A 11491 11510 CCAGGTCTTACTTTTCTTGA  85 177

TABLE 4 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 GFAP SEQ Compound Start Stop Start Stop RNA ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047146   58   77  3506  3525 CTCCTCTCCATCCTGCTCTG  94 178 1047162  138  157  3586  3605 GACGGCGGCCAGGAGCCAGG 115 179 1047178  274  293  3722  3741 AGCTCCATCATCTCTGCCCG 117 180 1047194  495  514  3943  3962 TGGCCAGGTCCTGTGCCAGA  67* 181 1047210  588  607  5177  5196 GGGTGGCTTCATCTGCTTCC  18* 182 1047226  656  675  5245  5264 GATCTTCCTCAAGAACCGGA  62 183 1047242  822  841  5708  5727 AGCGGTACCACTCTTCGGCT 113 184 1047258 1052 1071  7624  7643 CCCCTCTTCCTCCAGCCGCG  16 185 1047274 1253 1272 10867 10886 GCCTTCTGACACAGACTTGG 113 186 1047290 1309 1328 N/A N/A ATGACCTCTCCATCCCGCAT 108 187 1047306 1353 1372 11642 11661 TCCTGCCTCACATCACATCC  75 188 1047322 1499 1518 11788 11807 GCAAGCTGACCTAGGGACAG  68 189 1047338 1632 1651 11921 11940 TCCTCATTCTAACGCAAGCT  68 190 1047354 1666 1685 11955 11974 GGTGCCCCCCGCCCTCCTCC  90 191 1047370 1740 1759 12029 12048 CTCCGAGAGAACCTCCATCT  77 192 1047386 1793 1812 12082 12101 TCTCAGTTTTCCTCCAGCAG  33 193 1047402 1888 1907 12177 12196 AGCATAGGGATATCCCACCT  63 194 1047418 2063 2082 12352 12371 GGCAGGTGACTGCCCCAGGT 106 195 1047434 2182 2201 12471 12490 AAGTGCTGAGAATCAAGCTC 103 196 1047450 2239 2258 12528 12547 GCCCCCCTCTATCCCTCCCA 103 197 1047466 2329 2348 12618 12637 CTCCTCCATCTCTACCAGCG  52 198 1047482 2381 2400 12670 12689 ACTGGTCACCCACAACCCCT  79 199 1047498 2461 2480 12750 12769 CATCCCCTTTCTCTCCTGTT  74 200 1047514 2532 2551 12821 12840 CGGCCTGGTATGACACAGCA  91 201 1047530 2782 2801 13071 13090 GAGCACCCGGCCTCCAGGCT  80 202 1047546 2864 2883 13153 13172 CTGGGCACAGATCCCACCAG 107 203 1047562 2927 2946 13216 13235 GGACCCTTCTTCGGCCTTAG  69 204 1047578 3043 3062 13332 13351 TTATTTTTCCTCAGCGACTA  75 205 1047594 3062 3081 13351 13370 AAGGGCGCAGCATTTGTCTT  86 206 1047610 N/A N/A  8782  8801 GTGAGGCTTTTGAGATATCT  33 207 1047626 N/A N/A  9038  9057 TCTGCCAGTTTAATGTACAG 114 208 1047642 N/A N/A  9078  9097 CTGCGCACCCAAGGACTCAC  96 209 1047658 N/A N/A  9257  9276 CCTGAGGGAAGAATCCTCTG  86 210 1047674 N/A N/A  8390  8409 CCACGAGGCCCTGCTGTACT 111 211 1047690 N/A N/A  8461  8480 CCCTGATCCTCAGTCCCAGT  82 212 1047706 N/A N/A  8534  8553 CCCTGGTATGATAGGCTCTG  62 213 1047722 N/A N/A  8645  8664 AGGGCAGATGTCAAGCTCTC 110 214 1047738 N/A N/A  3968  3987 TCCCCTCCTCACTTCTGCCT  91 215 1047754 N/A N/A  4097  4116 GCAAGGATAGTGCCCCATCA  94 216 1047770 N/A N/A  4268  4287 CCACAGTCACAAAGCCCAGC  84 217 1047786 N/A N/A  4325  4344 TCCGCTTCCAACTCCTCCTT 107 218 1047802 N/A N/A  4362  4381 CCCCAGAATCCAATCTCCCT  84 219 1047818 N/A N/A  4477  4496 CCACCGCTTCACAGCTGTGC  77 220 1047834 N/A N/A  4528  4547 GGGATTCCTCTGATCCCAGG  93 221 1047850 N/A N/A  4706  4725 TGCCTTAACTCATTACTAAG  64 222 1047866 N/A N/A  4809  4828 ACAGAGACCACCCCCACCCA  63 223 1047882 N/A N/A  4959  4978 CTGACCTGTCTATAGGCAGC  89* 224 1047898 N/A N/A  5085  5104 GCCTTACCCCTCCTTCTGGG  100 225 1047914 N/A N/A  5268  5287 TGCCCTGGCCTCACCTCCTC  98 226 1047930 N/A N/A  5401  5420 GTCTTTCATTTCCTGTCTCT  55 227 1047946 N/A N/A  5562  5581 TCCCGAACCTCCTGACCAGG 117 228 1047962 N/A N/A  5803  5822 CCAGCTTCCTCCACCCTCCT 137 229 1047978 N/A N/A  5936  5955 TCAGCTACTACTAATAATAG  91 230 1047994 N/A N/A  6077  6096 AACTCTACCACTTAGGAGCT 130 231 1048010 N/A N/A  6148  6167 GTAACCCAAAACAGACTGGC  85 232 1048026 N/A N/A  6283  6302 CTCCCACACTACATATAAGC  82 233 1048042 N/A N/A  6331  6350 TGCCTGTCCTGCCTAGCCCA  72 234 1048058 N/A N/A  6467  6486 TCTGCCACTCACACTCCTCA  95 235 1048074 N/A N/A  6549  6568 TTCTAATAGCCCTTTCTCCC  83 236 1048090 N/A N/A  6990  7009 TCAGCAAGCGAATGAATGAA 157 237 1048106 N/A N/A  7075  7094 GCTGGGACTTTTCCCAACAA  91 238 1048122 N/A N/A  7449  7468 AGGCCCCGCCTCTAGCCCGG 110 239 1048138 N/A N/A  7920  7939 TCATCAAACATCTAGTGACT  91 240 1048154 N/A N/A  8112  8131 TCTATCTGAAGGAAGATGGA  90 241 1048170 N/A N/A  8329  8348 GTGATCCTGAAAGAAAGCAG  68 242 1048186 N/A N/A  9375  9394 TTTAGCTCCCCCCTCCCCCC 126 243 1048202 N/A N/A  9566  9585 TGCTTTAGTGACCTGTGACT  74 244 1048218 N/A N/A  9614  9633 CTTTCCTGTCCCCTTTCCTC 134 245 1048234 N/A N/A  9793  9812 AGCCACCAACCAGCCACATG  83 246 1048250 N/A N/A 10006 10025 CCTGTAATCCCCTTACTCGG  92 247 1048266 N/A N/A 10652 10671 GCTGCCAGAGTCCTGGCTGC  81 248 1048282 N/A N/A 10742 10761 CCATCATGAGTATGAGAACC  88 249 1048298 N/A N/A 10814 10833 GAGGCCTCTCATGGACTTTC  84 250 1048314 N/A N/A 10953 10972 AGCCAGAGCCTGACTGGGCC  80 251 1048330 N/A N/A 11080 11099 GGAATTACATTCAGTTTCCT  72 252 1048346 N/A N/A 11268 11287 CTCCCCATCCCCAACTGTGT 107 253 1048362 N/A N/A 11331 11350 CGCCAAATCCCCTCTTCCCA 106 254 1048378 N/A N/A 11492 11511 CCCAGGTCTTACTTTTCTTG  80 255

TABLE 5 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 GFAP SEQ Compound Start Stop Start Stop RNA ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047147   61   80  3509  3528 CGTCTCCTCTCCATCCTGCT  74 256 1047163  139  158  3587  3606 AGACGGCGGCCAGGAGCCAG 100 257 1047179  275  294  3723  3742 GAGCTCCATCATCTCTGCCC 116 258 1047195  500  519  3948  3967 CACAGTGGCCAGGTCCTGTG  70* 259 1047211  589  608  5178  5197 AGGGTGGCTTCATCTGCTTC  37* 260 1047227  657  676  5246  5265 GGATCTTCCTCAAGAACCGG  90 261 1047243  823  842  5709  5728 GAGCGGTACCACTCTTCGGC  83 262 1047259 1054 1073  7626  7645 TGCCCCTCTTCCTCCAGCCG  63 263 1047275 1254 1273 10868 10887 GGCCTTCTGACACAGACTTG 116 264 1047291 1311 1330 N/A N/A TAATGACCTCTCCATCCCGC  91 265 1047307 1354 1373 11643 11662 GTCCTGCCTCACATCACATC  88 266 1047323 1524 1543 11813 11832 CCTGATACTGACGGAGCCTA  56 267 1047339 1633 1652 11922 11941 CTCCTCATTCTAACGCAAGC 116 268 1047355 1668 1687 11957 11976 TAGGTGCCCCCCGCCCTCCT  68 269 1047371 1754 1773 12043 12062 ACAGTTCCCAGATACTCCGA  80 270 1047387 1794 1813 12083 12102 GTCTCAGTTTTCCTCCAGCA  12 271 1047403 1966 1985 12255 12274 CCAATCTATAATCCCAGCTA  89 272 1047419 2065 2084 12354 12373 TGGGCAGGTGACTGCCCCAG 110 273 1047435 2192 2211 12481 12500 CAGATCCCCCAAGTGCTGAG  87 274 1047451 2240 2259 12529 12548 AGCCCCCCTCTATCCCTCCC  97 275 1047467 2335 2354 12624 12643 TGCCTCCTCCTCCATCTCTA  72 276 1047483 2384 2403 12673 12692 GCAACTGGTCACCCACAACC  49 277 1047499 2462 2481 12751 12770 ACATCCCCTTTCTCTCCTGT  77 278 1047515 2675 2694 12964 12983 TTTGTGTGTGAGTAAGAAGG  49 279 1047531 2785 2804 13074 13093 CCTGAGCACCCGGCCTCCAG  80 280 1047547 2865 2884 13154 13173 TCTGGGCACAGATCCCACCA  85 281 1047563 2928 2947 13217 13236 AGGACCCTTCTTCGGCCTTA  70 282 1047579 3044 3063 13333 13352 TTTATTTTTCCTCAGCGACT  47 283 1047611 N/A N/A  8826  8845 TTCCATTTACAATCTGGTGA  93 285 1047627 N/A N/A  9040  9059 GCTCTGCCAGTTTAATGTAC 128 286 1047643 N/A N/A  9079  9098 ACTGCGCACCCAAGGACTCA  77 287 1047659 N/A N/A  9283  9302 ACTTTATTCACTGCAAGAGC  65 288 1047675 N/A N/A  8398  8417 TGCCCTTCCCACGAGGCCCT  53 289 1047691 N/A N/A  8462  8481 GCCCTGATCCTCAGTCCCAG  92 290 1047707 N/A N/A  8535  8554 ACCCTGGTATGATAGGCTCT  46 291 1047723 N/A N/A  8662  8681 CTCAGGGATCTGCAGACAGG  81 292 1047739 N/A N/A  3969  3988 ATCCCCTCCTCACTTCTGCC  85* 293 1047755 N/A N/A  4119  4138 GTCCCTCCCATCATGTTGGG  83 294 1047771 N/A N/A  4270  4289 GCCCACAGTCACAAAGCCCA  68 295 1047787 N/A N/A  4327  4346 TCTCCGCTTCCAACTCCTCC 109 296 1047803 N/A N/A  4363  4382 ACCCCAGAATCCAATCTCCC  81 297 1047819 N/A N/A  4503  4522 ACCTTTTGAAATGAATTTTA  65 298 1047835 N/A N/A  4588  4607 CTCCTGCACTTGAAGGCACA 101 299 1047851 N/A N/A  4707  4726 TTGCCTTAACTCATTACTAA  77 300 1047867 N/A N/A  4811  4830 TCACAGAGACCACCCCCACC  97 301 1047883 N/A N/A  4964  4983 CCTCCCTGACCTGTCTATAG 103* 302 1047899 N/A N/A  5086  5105 TGCCTTACCCCTCCTTCTGG  76 303 1047915 N/A N/A  5270  5289 TCTGCCCTGGCCTCACCTCC 102 304 1047931 N/A N/A  5403  5422 TTGTCTTTCATTTCCTGTCT  67 305 1047947 N/A N/A  5563  5582 TTCCCGAACCTCCTGACCAG 131 306 1047963 N/A N/A  5804  5823 CCCAGCTTCCTCCACCCTCC  58 307 1047979 N/A N/A  5937  5956 ATCAGCTACTACTAATAATA 128 308 1047995 N/A N/A  6078  6097 CAACTCTACCACTTAGGAGC  97 309 1048011 N/A N/A  6150  6169 CAGTAACCCAAAACAGACTG  85 310 1048027 N/A N/A  6284  6303 GCTCCCACACTACATATAAG  48 311 1048043 N/A N/A  6352  6371 CTTCTCTTCCTGTCCACAGC  93 312 1048059 N/A N/A  6471  6490 GGCTTCTGCCACTCACACTC  85 313 1048075 N/A N/A  6550  6569 GTTCTAATAGCCCTTTCTCC 124 314 1048091 N/A N/A  6991  7010 GTCAGCAAGCGAATGAATGA  85 315 1048107 N/A N/A  7109  7128 CAGCACCCCAGTTAACCCCA  73 316 1048123 N/A N/A  7450  7469 CAGGCCCCGCCTCTAGCCCG  88 317 1048139 N/A N/A  7928  7947 CCATTCAGTCATCAAACATC  76 318 1048155 N/A N/A  8120  8139 GGCGCATGTCTATCTGAAGG  76 319 1048171 N/A N/A  8330  8349 GGTGATCCTGAAAGAAAGCA 103 320 1048187 N/A N/A  9391  9410 TGCAAGTAAAAAGTAATTTA  69 321 1048203 N/A N/A  9568  9587 TGTGCTTTAGTGACCTGTGA  31 322 1048219 N/A N/A  9619  9638 GGTCCCTTTCCTGTCCCCTT  53 323 1048235 N/A N/A  9794  9813 TAGCCACCAACCAGCCACAT  97 324 1048251 N/A N/A 10007 10026 ACCTGTAATCCCCTTACTCG  89 325 1048267 N/A N/A 10657 10676 GTGCTGCTGCCAGAGTCCTG  35 326 1048283 N/A N/A 10750 10769 CCCCCTCCCCATCATGAGTA 100 327 1048299 N/A N/A 10841 10860 GGCTGGTTTCTGCAGATGTG  87 328 1048315 N/A N/A 10954 10973 CAGCCAGAGCCTGACTGGGC 188 329 1048331 N/A N/A 11086 11105 GGAAACGGAATTACATTCAG 130 330 1048347 N/A N/A 11269 11288 CCTCCCCATCCCCAACTGTG  99 331 1048363 N/A N/A 11332 11351 ACGCCAAATCCCCTCTTCCC  93 332 1048379 N/A N/A 11501 11520 CCCCGACTTCCCAGGTCTTA  91 333

TABLE 6 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 GFAP SEQ Compound Start Stop Start Stop RNA ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047148  112  131  3560  3579 ACCATCATCTCCCCTGAGGA 116 334 1047164  150  169  3598  3617 TGCCAGGACCCAGACGGCGG  96 335 1047180  277  296  3725  3744 TTGAGCTCCATCATCTCTGC 123 336 1047196  503  522  3951  3970 CCTCACAGTGGCCAGGTCCT  27* 337 1047212  615  634  5204  5223 TCTTCCTCTCCAGATCCAGA  77 338 1047228  658  677  5247  5266 TGGATCTTCCTCAAGAACCG  75 339 1047244  824  843  5710  5729 GGAGCGGTACCACTCTTCGG  74 340 1047260 1058 1077  7630  7649 GCTCTGCCCCTCTTCCTCCA  82 341 1047276 1255 1274 10869 10888 TGGCCTTCTGACACAGACTT 137 342 1047292 1312 1331 N/A N/A TTAATGACCTCTCCATCCCG  81 343 1047308 1356 1375 11645 11664 GGGTCCTGCCTCACATCACA  69 344 1047324 1526 1545 11815 11834 GGCCTGATACTGACGGAGCC  73 345 1047340 1634 1653 11923 11942 CCTCCTCATTCTAACGCAAG  63 346 1047356 1672 1691 11961 11980 GTAGTAGGTGCCCCCCGCCC  84 347 1047372 1755 1774 12044 12063 CACAGTTCCCAGATACTCCG  49 348 1047388 1795 1814 12084 12103 AGTCTCAGTTTTCCTCCAGC  24 349 1047404 1999 2018 12288 12307 GGAGAACAACCCTCTGAGCT  75 350 1047420 2101 2120 12390 12409 CACTTGAGTCATCGCTCAGG 107 351 1047436 2193 2212 12482 12501 ACAGATCCCCCAAGTGCTGA  73 352 1047452 2241 2260 12530 12549 CAGCCCCCCTCTATCCCTCC  88 353 1047468 2337 2356 12626 12645 ATTGCCTCCTCCTCCATCTC  93 354 1047484 2395 2414 12684 12703 AGAGGCCAAGTGCAACTGGT  80 355 1047500 2463 2482 12752 12771 TACATCCCCTTTCTCTCCTG  47 356 1047516 2699 2718 12988 13007 CACTACCTAGAATACTGGGT  92 357 1047532 2794 2813 13083 13102 CGTGTCAGCCCTGAGCACCC 129 358 1047548 2866 2885 13155 13174 CTCTGGGCACAGATCCCACC 141 359 1047564 2931 2950 13220 13239 GGAAGGACCCTTCTTCGGCC  67 360 1047580 3045 3064 13334 13353 CTTTATTTTTCCTCAGCGAC  35 361 1047612 N/A N/A  8827  8846 GTTCCATTTACAATCTGGTG  60 363 1047628 N/A N/A  9053  9072 TTACCACTAACAAGCTCTGC  76 364 1047644 N/A N/A  9081  9100 CCACTGCGCACCCAAGGACT  89 365 1047660 N/A N/A  9290  9309 ACATAAAACTTTATTCACTG  74 366 1047676 N/A N/A  8399  8418 GTGCCCTTCCCACGAGGCCC 176 367 1047692 N/A N/A  8463  8482 TGCCCTGATCCTCAGTCCCA  81 368 1047708 N/A N/A  8536  8555 TACCCTGGTATGATAGGCTC  60 369 1047724 N/A N/A  8669  8688 GTGCTTGCTCAGGGATCTGC  56 370 1047740 N/A N/A  3971  3990 CCATCCCCTCCTCACTTCTG  85* 371 1047756 N/A N/A  4120  4139 GGTCCCTCCCATCATGTTGG  76 372 1047772 N/A N/A  4272  4291 CTGCCCACAGTCACAAAGCC 103 373 1047788 N/A N/A  4328  4347 TTCTCCGCTTCCAACTCCTC 133 374 1047804 N/A N/A  4364  4383 CACCCCAGAATCCAATCTCC  78 375 1047820 N/A N/A  4507  4526 AACCACCTTTTGAAATGAAT 118 376 1047836 N/A N/A  4596  4615 CACATGTCCTCCTGCACTTG 108 377 1047852 N/A N/A  4708  4727 TTTGCCTTAACTCATTACTA  91 378 1047868 N/A N/A  4812  4831 GTCACAGAGACCACCCCCAC 108 379 1047884 N/A N/A  4965  4984 ACCTCCCTGACCTGTCTATA  83 380 1047900 N/A N/A  5087  5106 TTGCCTTACCCCTCCTTCTG 102 381 1047916 N/A N/A  5282  5301 AGCTTTCCTCCCTCTGCCCT  92 382 1047932 N/A N/A  5405  5424 GTTTGTCTTTCATTTCCTGT  82 383 1047948 N/A N/A  5564  5583 GTTCCCGAACCTCCTGACCA 103 384 1047964 N/A N/A  5805  5824 TCCCAGCTTCCTCCACCCTC  78 385 1047980 N/A N/A  5938  5957 TATCAGCTACTACTAATAAT 106 386 1047996 N/A N/A  6079  6098 CCAACTCTACCACTTAGGAG  96 387 1048012 N/A N/A  6151  6170 TCAGTAACCCAAAACAGACT 124 388 1048028 N/A N/A  6285  6304 GGCTCCCACACTACATATAA 134 389 1048044 N/A N/A  6353  6372 TCTTCTCTTCCTGTCCACAG  93 390 1048060 N/A N/A  6473  6492 GTGGCTTCTGCCACTCACAC 129 391 1048076 N/A N/A  6556  6575 CCCTGGGTTCTAATAGCCCT  89 392 1048092 N/A N/A  6993  7012 TGGTCAGCAAGCGAATGAAT  81 393 1048108 N/A N/A  7111  7130 AGCAGCACCCCAGTTAACCC 104 394 1048124 N/A N/A  7451  7470 CCAGGCCCCGCCTCTAGCCC  89 395 1048140 N/A N/A  7932  7951 CCATCCATTCAGTCATCAAA  70 396 1048156 N/A N/A  8142  8161 GGCTTGAGTGTTATCTGGGA  74 397 1048172 N/A N/A  8332  8351 ATGGTGATCCTGAAAGAAAG  87 398 1048188 N/A N/A  9392  9411 ATGCAAGTAAAAAGTAATTT  61 399 1048204 N/A N/A  9581  9600 TCTGCCATTTATCTGTGCTT  61 400 1048220 N/A N/A  9621  9640 TAGGTCCCTTTCCTGTCCCC  60 401 1048236 N/A N/A  9795  9814 TTAGCCACCAACCAGCCACA 106 402 1048252 N/A N/A 10009 10028 GCACCTGTAATCCCCTTACT  76 403 1048268 N/A N/A 10658 10677 AGTGCTGCTGCCAGAGTCCT  80 404 1048284 N/A N/A 10751 10770 CCCCCCTCCCCATCATGAGT  82 405 1048300 N/A N/A 10921 10940 TACCTCTCCATCCCGCATCT 109 406 1048316 N/A N/A 10976 10995 TGGCCTTGAGAATCCCTGGG  99 407 1048332 N/A N/A 11090 11109 CTGAGGAAACGGAATTACAT  83 408 1048348 N/A N/A 11272 11291 AGCCCTCCCCATCCCCAACT 121 409 1048364 N/A N/A 11333 11352 TACGCCAAATCCCCTCTTCC  92 410 1048380 N/A N/A 11504 11523 AGTCCCCGACTTCCCAGGTC 116 411

TABLE 7 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 GFAP SEQ Compound Start Stop Start Stop RNA ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047149  113  132  3561  3580 CACCATCATCTCCCCTGAGG 102 412 1047165  151  170  3599  3618 GTGCCAGGACCCAGACGGCG 120 413 1047181  279  298  3727  3746 CATTGAGCTCCATCATCTCT  92 414 1047197  525  544 N/A N/A TGGTTTCATCCTGGAGCTTC  39* 415 1047213  617  636  5206  5225 AATCTTCCTCTCCAGATCCA  79 416 1047229  659  678  5248  5267 GTGGATCTTCCTCAAGAACC  83 417 1047245  825  844  5711  5730 TGGAGCGGTACCACTCTTCG  77 418 1047261 1060 1079  7632  7651 AGGCTCTGCCCCTCTTCCTC  78 419 1047277 1257 1276 10871 10890 GGTGGCCTTCTGACACAGAC  98 420 1047293 1313 1332 N/A N/A CTTAATGACCTCTCCATCCC 105 421 1047309 1357 1376 11646 11665 TGGGTCCTGCCTCACATCAC 154 422 1047325 1529 1548 11818 11837 GCAGGCCTGATACTGACGGA  46 423 1047341 1635 1654 11924 11943 TCCTCCTCATTCTAACGCAA  53 424 1047357 1684 1703 11973 11992 GTGGAGGGCGATGTAGTAGG  42 425 1047373 1756 1775 12045 12064 GCACAGTTCCCAGATACTCC  33 426 1047389 1812 1831 12101 12120 CTTCCCTTTCCTGTCTGAGT  64 427 1047405 2003 2022 12292 12311 TCTAGGAGAACAACCCTCTG 160 428 1047421 2103 2122 12392 12411 GACACTTGAGTCATCGCTCA  63 429 1047437 2194 2213 12483 12502 AACAGATCCCCCAAGTGCTG  85 430 1047453 2242 2261 12531 12550 GCAGCCCCCCTCTATCCCTC 173 431 1047469 2341 2360 12630 12649 CCCAATTGCCTCCTCCTCCA  53 432 1047485 2407 2426 12696 12715 TTCCCACAATCCAGAGGCCA 101 433 1047501 2464 2483 12753 12772 ATACATCCCCTTTCTCTCCT  63 434 1047517 2701 2720 12990 13009 GGCACTACCTAGAATACTGG  73 435 1047533 2833 2852 13122 13141 GTCTGCTCAGTCAAAGCAGA  93 436 1047549 2877 2896 13166 13185 CCCAGTCCCATCTCTGGGCA 134 437 1047565 2932 2951 13221 13240 GGGAAGGACCCTTCTTCGGC  66 438 1047581 3046 3065 13335 13354 TCTTTATTTTTCCTCAGCGA  41 439 1047613 N/A N/A  8828  8847 CGTTCCATTTACAATCTGGT  47 441 1047629 N/A N/A  9056  9075 CCTTTACCACTAACAAGCTC 161 442 1047645 N/A N/A  9086  9105 CAGCTCCACTGCGCACCCAA  85 443 1047661 N/A N/A  9300  9319 AGAGCAGGGAACATAAAACT  69 444 1047677 N/A N/A  8400  8419 AGTGCCCTTCCCACGAGGCC  85 445 1047693 N/A N/A  8464  8483 TTGCCCTGATCCTCAGTCCC 117 446 1047709 N/A N/A  8547  8566 CCACCTAGAAGTACCCTGGT  75 447 1047725 N/A N/A  8688  8707 GAAAACACTCAGAAGGGCAG 101 448 1047741 N/A N/A  3972  3991 CCCATCCCCTCCTCACTTCT 133 449 1047757 N/A N/A  4122  4141 CTGGTCCCTCCCATCATGTT 136 450 1047773 N/A N/A  4273  4292 GCTGCCCACAGTCACAAAGC 126 451 1047789 N/A N/A  4334  4353 TCAACCTTCTCCGCTTCCAA  66 452 1047805 N/A N/A  4369  4388 TTCTTCACCCCAGAATCCAA 123 453 1047821 N/A N/A  4508  4527 TAACCACCTTTTGAAATGAA  75 454 1047837 N/A N/A  4657  4676 CTGCTCACACAGGCGCATCC  97 455 1047853 N/A N/A  4709  4728 TTTTGCCTTAACTCATTACT 142 456 1047869 N/A N/A  4813  4832 TGTCACAGAGACCACCCCCA  87 457 1047885 N/A N/A  4968  4987 TCCACCTCCCTGACCTGTCT  84 458 1047901 N/A N/A  5090  5109 GCCTTGCCTTACCCCTCCTT  86 459 1047917 N/A N/A  5284  5303 TGAGCTTTCCTCCCTCTGCC  93 460 1047933 N/A N/A  5417  5436 TAGTGTCTTTCTGTTTGTCT  82 461 1047949 N/A N/A  5565  5584 AGTTCCCGAACCTCCTGACC  74 462 1047965 N/A N/A  5806  5825 CTCCCAGCTTCCTCCACCCT 101 463 1047981 N/A N/A  5939  5958 GTATCAGCTACTACTAATAA 137 464 1047997 N/A N/A  6080  6099 TCCAACTCTACCACTTAGGA 112 465 1048013 N/A N/A  6152  6171 CTCAGTAACCCAAAACAGAC  79 466 1048029 N/A N/A  6287  6306 CTGGCTCCCACACTACATAT  86 467 1048045 N/A N/A  6354  6373 TTCTTCTCTTCCTGTCCACA  92 468 1048061 N/A N/A  6474  6493 AGTGGCTTCTGCCACTCACA  88 469 1048077 N/A N/A  6557  6576 ACCCTGGGTTCTAATAGCCC 104 470 1048093 N/A N/A  7008  7027 ACCTAGCACAACACCTGGTC  77 471 1048109 N/A N/A  7318  7337 GCGCTCACCGTGCCGCGCAG  85 472 1048125 N/A N/A  7497  7516 GAGCCCCGACCCGACTTGGG  55 473 1048141 N/A N/A  7943  7962 GTTGAATCCATCCATCCATT  73 474 1048157 N/A N/A  8195  8214 AGCTTTTTCCCCAGCAGCCA  91 475 1048173 N/A N/A  8333  8352 AATGGTGATCCTGAAAGAAA  80 476 1048189 N/A N/A  9415  9434 AGCTAAGAATCATTTCAGGG  77 477 1048205 N/A N/A  9584  9603 CTCTCTGCCATTTATCTGTG  67 478 1048221 N/A N/A  9622  9641 ATAGGTCCCTTTCCTGTCCC  67 479 1048237 N/A N/A  9796  9815 CTTAGCCACCAACCAGCCAC 123 480 1048253 N/A N/A 10010 10029 CGCACCTGTAATCCCCTTAC  97 481 1048269 N/A N/A 10666 10685 ATCCCAATAGTGCTGCTGCC  79 482 1048285 N/A N/A 10755 10774 CGCACCCCCCTCCCCATCAT 109 483 1048301 N/A N/A 10925 10944 TCCTTACCTCTCCATCCCGC 106 484 1048317 N/A N/A 10993 11012 GGCTTTCCTCCATGGCCTGG  93 485 1048333 N/A N/A 11092 11111 GACTGAGGAAACGGAATTAC  86 486 1048349 N/A N/A 11278 11297 ATGGAAAGCCCTCCCCATCC  86 487 1048365 N/A N/A 11334 11353 ATACGCCAAATCCCCTCTTC 105 488 1048381 N/A N/A 11505 11524 AAGTCCCCGACTTCCCAGGT  79 489

TABLE 8 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047150  115  134  3563  3582 CCCACCATCATCTCCCCTGA  93 490 1047166  156  175  3604  3623 GGCGGGTGCCAGGACCCAGA  79 491 1047182  358  377  3806  3825 AGCTGGTTCAGCTCAGCAGC 114 492 1047198  528  547  4913  4932 GGTTGGTTTCATCCTGGAGC  19* 493 1047214  619  638  5208  5227 TCAATCTTCCTCTCCAGATC 115 494 1047230  680  699 N/A N/A GAGTTCCCGAACCTCCTCCT  85 495 1047246  838  857 N/A N/A AGGTCTGCAAACTTGGAGCG  90 496 1047262 1064 1083  7636  7655 CTTGAGGCTCTGCCCCTCTT  88 497 1047278 1258 1277 10872 10891 AGGTGGCCTTCTGACACAGA  94 498 1047294 1314 1333 N/A N/A CCTTAATGACCTCTCCATCC 154 499 1047310 1358 1377 11647 11666 GTGGGTCCTGCCTCACATCA  88 500 1047326 1530 1549 11819 11838 GGCAGGCCTGATACTGACGG 103 501 1047342 1636 1655 11925 11944 TTCCTCCTCATTCTAACGCA  66 502 1047358 1685 1704 11974 11993 TGTGGAGGGCGATGTAGTAG  50 503 1047374 1757 1776 12046 12065 GGCACAGTTCCCAGATACTC  40 504 1047390 1813 1832 12102 12121 CCTTCCCTTTCCTGTCTGAG  81 505 1047406 2004 2023 12293 12312 GTCTAGGAGAACAACCCTCT  96 506 1047422 2112 2131 12401 12420 GTGGACTGAGACACTTGAGT  54 507 1047438 2201 2220 12490 12509 CGTACACAACAGATCCCCCA  61 508 1047454 2243 2262 12532 12551 GGCAGCCCCCCTCTATCCCT  57 509 1047470 2342 2361 12631 12650 TCCCAATTGCCTCCTCCTCC  66 510 1047486 2413 2432 12702 12721 CCTTAATTCCCACAATCCAG 147 511 1047502 2465 2484 12754 12773 GATACATCCCCTTTCTCTCC  67 512 1047518 2702 2721 12991 13010 GGGCACTACCTAGAATACTG  43 513 1047534 2843 2862 13132 13151 CTGCTCACCAGTCTGCTCAG  97 514 1047550 2878 2897 13167 13186 TCCCAGTCCCATCTCTGGGC 106 515 1047566 2933 2952 13222 13241 AGGGAAGGACCCTTCTTCGG  71 516 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG   9 517 1047614 N/A N/A  8850  8869 GCAGCTAACCGCGAGCCGGC 132 519 1047630 N/A N/A  9057  9076 ACCTTTACCACTAACAAGCT  89 520 1047646 N/A N/A  9088  9107 AGCAGCTCCACTGCGCACCC 116 521 1047662 N/A N/A  9311  9330 ATTTAACATTAAGAGCAGGG  45 522 1047678 N/A N/A  8401  8420 CAGTGCCCTTCCCACGAGGC 110 523 1047694 N/A N/A  8467  8486 CCTTTGCCCTGATCCTCAGT  77 524 1047710 N/A N/A  8549  8568 CCCCACCTAGAAGTACCCTG  99 525 1047726 N/A N/A  8689  8708 AGAAAACACTCAGAAGGGCA 169 526 1047742 N/A N/A  3973  3992 CCCCATCCCCTCCTCACTTC 100 527 1047758 N/A N/A  4124  4143 TTCTGGTCCCTCCCATCATG  77 528 1047774 N/A N/A  4277  4296 GCTCGCTGCCCACAGTCACA 123 529 1047790 N/A N/A  4338  4357 GACATCAACCTTCTCCGCTT  67 530 1047806 N/A N/A  4375  4394 CTCACTTTCTTCACCCCAGA 156 531 1047822 N/A N/A  4510  4529 GGTAACCACCTTTTGAAATG  91 532 1047838 N/A N/A  4659  4678 TTCTGCTCACACAGGCGCAT 148 533 1047854 N/A N/A  4712  4731 GGCTTTTGCCTTAACTCATT  72 534 1047870 N/A N/A  4815  4834 GCTGTCACAGAGACCACCCC  93 535 1047886 N/A N/A  4971  4990 CCCTCCACCTCCCTGACCTG  59 536 1047902 N/A N/A  5092  5111 CAGCCTTGCCTTACCCCTCC  70 537 1047918 N/A N/A  5285  5304 TTGAGCTTTCCTCCCTCTGC  80 538 1047934 N/A N/A  5429  5448 TTCCGTCTCCCTTAGTGTCT 105 539 1047950 N/A N/A  5566  5585 GAGTTCCCGAACCTCCTGAC  70 540 1047966 N/A N/A  5813  5832 GGATATTCTCCCAGCTTCCT  90 541 1047982 N/A N/A  5945  5964 AGAACAGTATCAGCTACTAC 106 542 1047998 N/A N/A  6085  6104 GGAAATCCAACTCTACCACT 106 543 1048014 N/A N/A  6153  6172 GCTCAGTAACCCAAAACAGA  96 544 1048030 N/A N/A  6288  6307 CCTGGCTCCCACACTACATA 116 545 1048046 N/A N/A  6356  6375 CCTTCTTCTCTTCCTGTCCA 133 546 1048062 N/A N/A  6486  6505 TGCTCAGACACCAGTGGCTT 109 547 1048078 N/A N/A  6567  6586 GCCTGGCCTCACCCTGGGTT  88 548 1048094 N/A N/A  7023  7042 CTGCCAGACCTCAGCACCTA  81 549 1048110 N/A N/A  7322  7341 GGCCGCGCTCACCGTGCCGC 109 550 1048126 N/A N/A  7498  7517 GGAGCCCCGACCCGACTTGG 124 551 1048142 N/A N/A  7944  7963 GGTTGAATCCATCCATCCAT 105 552 1048158 N/A N/A  8196  8215 TAGCTTTTTCCCCAGCAGCC  89 553 1048174 N/A N/A  8334  8353 GAATGGTGATCCTGAAAGAA  81 554 1048190 N/A N/A  9462  9481 AGTGGTCCTAAATATTCTAG  66 555 1048206 N/A N/A  9585  9604 TCTCTCTGCCATTTATCTGT  91 556 1048222 N/A N/A  9623  9642 CATAGGTCCCTTTCCTGTCC  71 557 1048238 N/A N/A  9799  9818 CAACTTAGCCACCAACCAGC 120 558 1048254 N/A N/A 10576 10595 GGCTTTCTGAAAACCCAGCA  72 559 1048270 N/A N/A 10673 10692 CCCCCAAATCCCAATAGTGC 117 560 1048286 N/A N/A 10756 10775 TCGCACCCCCCTCCCCATCA 106 561 1048302 N/A N/A 10926 10945 CTCCTTACCTCTCCATCCCG 120 562 1048318 N/A N/A 10994 11013 AGGCTTTCCTCCATGGCCTG  91 563 1048334 N/A N/A 11110 11129 TAGAACAGCCTATGGAGGGA  52 564 1048350 N/A N/A 11300 11319 GTTTCCTTTTACCAAGCTGG  34 565 1048366 N/A N/A 11335 11354 GATACGCCAAATCCCCTCTT  94 566 1048382 N/A N/A 11508 11527 GGGAAGTCCCCGACTTCCCA  92 567

TABLE 9 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047151  116  135  3564  3583 CCCCACCATCATCTCCCCTG  89 568 1047167  173  192  3621  3640 CATTCGAGCCAGGGAGAGGC  82 569 1047183  372  391  3820  3839 GCTCCTTGGCCCGCAGCTGG 147 570 1047199  529  548  4914  4933 AGGTTGGTTTCATCCTGGAG  47* 571 1047215  620  639  5209  5228 CTCAATCTTCCTCTCCAGAT  93 572 1047231  683  702  5569  5588 CTGGAGTTCCCGAACCTCCT 113 573 1047247  943  962  7300  7319 AGAGACTCCAGGTCGCAGGT 104 574 1047263 1098 1117  7670  7689 CCTGGTACTCCTGCAAGTGG 139 575 1047279 1260 1279 10874 10893 TGAGGTGGCCTTCTGACACA 106 576 1047295 1319 1338 N/A N/A GGACTCCTTAATGACCTCTC  77 577 1047311 1370 1389 11659 11678 AGAGGCCACCAGGTGGGTCC  88 578 1047327 1531 1550 11820 11839 TGGCAGGCCTGATACTGACG  71 579 1047343 1637 1656 11926 11945 CTTCCTCCTCATTCTAACGC  68 580 1047359 1714 1733 12003 12022 ACAGTTTCCATAACAACAGG 137 581 1047375 1760 1779 12049 12068 AAAGGCACAGTTCCCAGATA 106 582 1047391 1814 1833 12103 12122 GCCTTCCCTTTCCTGTCTGA  34 583 1047407 2005 2024 12294 12313 AGTCTAGGAGAACAACCCTC 113 584 1047423 2138 2157 12427 12446 GATGGCATCCCTGGATGGCA 113 585 1047439 2219 2238 12508 12527 GCACCTCATCCCTCTCCACG  75 586 1047455 2244 2263 12533 12552 AGGCAGCCCCCCTCTATCCC  77 587 1047471 2343 2362 12632 12651 ATCCCAATTGCCTCCTCCTC  96 588 1047487 2414 2433 12703 12722 TCCTTAATTCCCACAATCCA 105 589 1047503 2466 2485 12755 12774 GGATACATCCCCTTTCTCTC  48 590 1047519 2727 2746 13016 13035 GCCTCAGTTTTACAATTGTA  90 591 1047535 2844 2863 13133 13152 TCTGCTCACCAGTCTGCTCA  92 592 1047551 2880 2899 13169 13188 CCTCCCAGTCCCATCTCTGG  90 593 1047567 2937 2956 13226 13245 GGAGAGGGAAGGACCCTTCT 105 594 1047583 3048 3067 13337 13356 TGTCTTTATTTTTCCTCAGC  10 595 1047615 N/A N/A  8851  8870 GGCAGCTAACCGCGAGCCGG  92 597 1047631 N/A N/A  9058  9077 CACCTTTACCACTAACAAGC 105 598 1047647 N/A N/A  9089  9108 GAGCAGCTCCACTGCGCACC  87 599 1047663 N/A N/A  9312  9331 TATTTAACATTAAGAGCAGG 106 600 1047679 N/A N/A  8402  8421 CCAGTGCCCTTCCCACGAGG  78 601 1047695 N/A N/A  8469  8488 TCCCTTTGCCCTGATCCTCA  74 602 1047711 N/A N/A  8551  8570 AGCCCCACCTAGAAGTACCC  86 603 1047727 N/A N/A  8690  8709 CAGAAAACACTCAGAAGGGC  93 604 1047743 N/A N/A  3974  3993 TCCCCATCCCCTCCTCACTT  92 605 1047759 N/A N/A  4126  4145 GTTTCTGGTCCCTCCCATCA  98 606 1047775 N/A N/A  4278  4297 AGCTCGCTGCCCACAGTCAC 170 607 1047791 N/A N/A  4339  4358 GGACATCAACCTTCTCCGCT  98 608 1047807 N/A N/A  4376  4395 CCTCACTTTCTTCACCCCAG 104 609 1047823 N/A N/A  4512  4531 CAGGTAACCACCTTTTGAAA  82 610 1047839 N/A N/A  4661  4680 GCTTCTGCTCACACAGGCGC  96 611 1047855 N/A N/A  4713  4732 GGGCTTTTGCCTTAACTCAT  89 612 1047871 N/A N/A  4833  4852 TCAGTCTCCCTTGAGGCAGC  87 613 1047887 N/A N/A  4972  4991 CCCCTCCACCTCCCTGACCT 109 614 1047903 N/A N/A  5093  5112 TCAGCCTTGCCTTACCCCTC  96 615 1047919 N/A N/A  5286  5305 GTTGAGCTTTCCTCCCTCTG  82 616 1047935 N/A N/A  5434  5453 TCTCTTTCCGTCTCCCTTAG  83 617 1047951 N/A N/A  5724  5743 GGCAGGGCTACCTTGGAGCG  98 618 1047967 N/A N/A  5814  5833 AGGATATTCTCCCAGCTTCC  74 619 1047983 N/A N/A  5946  5965 CAGAACAGTATCAGCTACTA  76 620 1047999 N/A N/A  6086  6105 TGGAAATCCAACTCTACCAC 122 621 1048015 N/A N/A  6154  6173 GGCTCAGTAACCCAAAACAG  82 622 1048031 N/A N/A  6290  6309 TTCCTGGCTCCCACACTACA 106 623 1048047 N/A N/A  6362  6381 GCCCTCCCTTCTTCTCTTCC  84 624 1048063 N/A N/A  6487  6506 CTGCTCAGACACCAGTGGCT 111 625 1048079 N/A N/A  6569  6588 TCGCCTGGCCTCACCCTGGG 101 626 1048095 N/A N/A  7024  7043 GCTGCCAGACCTCAGCACCT 192 627 1048111 N/A N/A  7328  7347 TGCCCTGGCCGCGCTCACCG  82 628 1048127 N/A N/A  7502  7521 CCGCGGAGCCCCGACCCGAC  90 629 1048143 N/A N/A  7945  7964 TGGTTGAATCCATCCATCCA 117 630 1048159 N/A N/A  8197  8216 CTAGCTTTTTCCCCAGCAGC 100 631 1048175 N/A N/A  9316  9335 CTAATATTTAACATTAAGAG 128 632 1048191 N/A N/A  9463  9482 TAGTGGTCCTAAATATTCTA 106 633 1048207 N/A N/A  9586  9605 TTCTCTCTGCCATTTATCTG  70 634 1048223 N/A N/A  9625  9644 CACATAGGTCCCTTTCCTGT  74 635 1048239 N/A N/A  9800  9819 CCAACTTAGCCACCAACCAG  96 636 1048255 N/A N/A 10577 10596 TGGCTTTCTGAAAACCCAGC  83 637 1048271 N/A N/A 10674 10693 GCCCCCAAATCCCAATAGTG 116 638 1048287 N/A N/A 10757 10776 ATCGCACCCCCCTCCCCATC 142 639 1048303 N/A N/A 10928 10947 CCCTCCTTACCTCTCCATCC 117 640 1048319 N/A N/A 10996 11015 CCAGGCTTTCCTCCATGGCC  88 641 1048335 N/A N/A 11111 11130 TTAGAACAGCCTATGGAGGG 115 642 1048351 N/A N/A 11301 11320 AGTTTCCTTTTACCAAGCTG  54 643 1048367 N/A N/A 11337 11356 CGGATACGCCAAATCCCCTC 101 644 1048383 N/A N/A 11539 11558 CAGGTCCACCACCACGAGGC 138 645

TABLE 10 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047152  117  136  3565  3584 CCCCCACCATCATCTCCCCT 104 646 1047168  175  194  3623  3642 GGCATTCGAGCCAGGGAGAG  90 647 1047184  435  454  3883  3902 GTTGATCGAGCCGCAGCCGC 104 648 1047200  530  549  4915  4934 CAGGTTGGTTTCATCCTGGA  56* 649 1047216  621  640  5210  5229 ACTCAATCTTCCTCTCCAGA  95 650 1047232  684  703  5570  5589 CCTGGAGTTCCCGAACCTCC 126 651 1047248  946  965  7303  7322 CGCAGAGACTCCAGGTCGCA 104 652 1047264 1100 1119  7672  7691 GTCCTGGTACTCCTGCAAGT  89 653 1047280 1273 1292 10887 10906 ACGATGTTCCTCTTGAGGTG  96 654 1047296 1320 1339 N/A N/A TGGACTCCTTAATGACCTCT 101 655 1047312 1372 1391 11661 11680 GCAGAGGCCACCAGGTGGGT  64 656 1047328 1580 1599 11869 11888 GGTGAGTTTCTTGTTAGTTG  29 657 1047344 1638 1657 11927 11946 CCTTCCTCCTCATTCTAACG  73 658 1047360 1715 1734 12004 12023 AACAGTTTCCATAACAACAG 115 659 1047376 1761 1780 12050 12069 CAAAGGCACAGTTCCCAGAT  94 660 1047392 1815 1834 12104 12123 GGCCTTCCCTTTCCTGTCTG 104 661 1047408 2021 2040 12310 12329 TAGACTGATCAGGGTCAGTC 125 662 1047424 2148 2167 12437 12456 CGTGCCCACAGATGGCATCC  87 663 1047440 2222 2241 12511 12530 CCAGCACCTCATCCCTCTCC 111 664 1047456 2246 2265 12535 12554 CCAGGCAGCCCCCCTCTATC  87 665 1047472 2345 2364 12634 12653 CCATCCCAATTGCCTCCTCC  76 666 1047488 2417 2436 12706 12725 ACTTCCTTAATTCCCACAAT 103 667 1047504 2468 2487 12757 12776 ATGGATACATCCCCTTTCTC  73 668 1047520 2728 2747 13017 13036 TGCCTCAGTTTTACAATTGT 109 669 1047536 2845 2864 13134 13153 GTCTGCTCACCAGTCTGCTC 110 670 1047552 2883 2902 13172 13191 GGCCCTCCCAGTCCCATCTC  85 671 1047568 2956 2975 13245 13264 AAAGGACACCAAGTCTTGGG  69 672 1047584 3049 3068 13338 13357 TTGTCTTTATTTTTCCTCAG  11 673 1047616 N/A N/A  8852  8871 AGGCAGCTAACCGCGAGCCG  94 675 1047632 N/A N/A  9059  9078 CCACCTTTACCACTAACAAG  89 676 1047648 N/A N/A  9101  9120 TCAGAGGCCCCAGAGCAGCT  86 677 1047664 N/A N/A  9314  9333 AATATTTAACATTAAGAGCA 105 678 1047680 N/A N/A  8404  8423 CTCCAGTGCCCTTCCCACGA  86 679 1047696 N/A N/A  8471  8490 GATCCCTTTGCCCTGATCCT  76 680 1047712 N/A N/A  8554  8573 GCAAGCCCCACCTAGAAGTA  76 681 1047728 N/A N/A  8691  8710 ACAGAAAACACTCAGAAGGG  92 682 1047744 N/A N/A  3984  4003 AGGCCCCCCTTCCCCATCCC  79 683 1047760 N/A N/A  4138  4157 GGCCCTGGGCCTGTTTCTGG  86 684 1047776 N/A N/A  4279  4298 GAGCTCGCTGCCCACAGTCA 119 685 1047792 N/A N/A  4340  4359 TGGACATCAACCTTCTCCGC 102 686 1047808 N/A N/A  4377  4396 CCCTCACTTTCTTCACCCCA  71 687 1047824 N/A N/A  4513  4532 CCAGGTAACCACCTTTTGAA 104 688 1047840 N/A N/A  4686  4705 GTGCCTTATCAGGGTTGGTG  64 689 1047856 N/A N/A  4714  4733 TGGGCTTTTGCCTTAACTCA  79 690 1047872 N/A N/A  4835  4854 CCTCAGTCTCCCTTGAGGCA 104 691 1047888 N/A N/A  4976  4995 CCCTCCCCTCCACCTCCCTG  79 692 1047904 N/A N/A  5094  5113 CTCAGCCTTGCCTTACCCCT 121 693 1047920 N/A N/A  5313  5332 TCTCCCTCTCTCAGTTGCAA  80 694 1047936 N/A N/A  5436  5455 TGTCTCTTTCCGTCTCCCTT  76 695 1047952 N/A N/A  5738  5757 CAGGCTGGCCCACAGGCAGG  96 696 1047968 N/A N/A  5815  5834 GAGGATATTCTCCCAGCTTC  83 697 1047984 N/A N/A  5993  6012 CACCTACTTCATAGTAAGGT 144 698 1048000 N/A N/A  6088  6107 GTTGGAAATCCAACTCTACC  75 699 1048016 N/A N/A  6155  6174 AGGCTCAGTAACCCAAAACA  97 700 1048032 N/A N/A  6298  6317 CAGTGTCTTTCCTGGCTCCC  72 701 1048048 N/A N/A  6363  6382 GGCCCTCCCTTCTTCTCTTC 106 702 1048064 N/A N/A  6491  6510 CACCCTGCTCAGACACCAGT 112 703 1048080 N/A N/A  6570  6589 CTCGCCTGGCCTCACCCTGG  80 704 1048096 N/A N/A  7028  7047 GCGGGCTGCCAGACCTCAGC  82 705 1048112 N/A N/A  7335  7354 CCCGTCCTGCCCTGGCCGCG  92 706 1048128 N/A N/A  7833  7852 AAAAAGACTCAGTCCCTGAA 103 707 1048144 N/A N/A  7946  7965 TTGGTTGAATCCATCCATCC  68 708 1048160 N/A N/A  8198  8217 CCTAGCTTTTTCCCCAGCAG  71 709 1048176 N/A N/A  9317  9336 ACTAATATTTAACATTAAGA  69 710 1048192 N/A N/A  9465  9484 TCTAGTGGTCCTAAATATTC 108 711 1048208 N/A N/A  9593  9612 TTCCTACTTCTCTCTGCCAT  80 712 1048224 N/A N/A  9633  9652 GCTCAATACACATAGGTCCC  82 713 1048240 N/A N/A  9801  9820 CCCAACTTAGCCACCAACCA 120 714 1048256 N/A N/A 10578 10597 CTGGCTTTCTGAAAACCCAG 119 715 1048272 N/A N/A 10675 10694 AGCCCCCAAATCCCAATAGT 116 716 1048288 N/A N/A 10759 10778 CCATCGCACCCCCCTCCCCA  88 717 1048304 N/A N/A 10929 10948 TCCCTCCTTACCTCTCCATC  73 718 1048320 N/A N/A 10997 11016 CCCAGGCTTTCCTCCATGGC 113 719 1048336 N/A N/A 11112 11131 CTTAGAACAGCCTATGGAGG  74 720 1048352 N/A N/A 11302 11321 TAGTTTCCTTTTACCAAGCT  97 721 1048368 N/A N/A 11338 11357 GCGGATACGCCAAATCCCCT  74 722 1048384 N/A N/A 11541 11560 CCCAGGTCCACCACCACGAG 105 723

TABLE 11 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047153  119  138  3567  3586 GCCCCCCACCATCATCTCCC  88 724 1047169  210  229  3658  3677 CCAGGGAGAAATCCACCCGG  81 725 1047185  440  459  3888  3907 GGTGAGTTGATCGAGCCGCA  70 726 1047201  539  558  4924  4943 TTCCAGCCTCAGGTTGGTTT  29* 727 1047217  627  646  5216  5235 CCAGCGACTCAATCTTCCTC  99 728 1047233  703  722  5589  5608 TGCTGTCGGGCCAGCTGCTC 118 729 1047249  990 1009  7562  7581 CCTCCTGCTCGCGCATCTGC  93 730 1047265 1101 1120  7673  7692 GGTCCTGGTACTCCTGCAAG 129 731 1047281 1274 1293 10888 10907 CACGATGTTCCTCTTGAGGT 104 732 1047297 1321 1340 N/A N/A TTGGACTCCTTAATGACCTC 110 733 1047313 1393 1412 11682 11701 TCGGGCCCCTCATGAGACGG  99 734 1047329 1605 1624 11894 11913 ATGCCCCTCCAGACTGCCCC 109 735 1047345 1640 1659 11929 11948 CTCCTTCCTCCTCATTCTAA  89 736 1047361 1716 1735 12005 12024 CAACAGTTTCCATAACAACA  62 737 1047377 1770 1789 12059 12078 GAGGAAACTCAAAGGCACAG  82 738 1047393 1816 1835 12105 12124 GGGCCTTCCCTTTCCTGTCT  64 739 1047409 2024 2043 12313 12332 TCTTAGACTGATCAGGGTCA 124 740 1047425 2166 2185 12455 12474 GCTCCCACCTGCCCACAGCG  79 741 1047441 2224 2243 12513 12532 TCCCAGCACCTCATCCCTCT 109 742 1047457 2247 2266 12536 12555 GCCAGGCAGCCCCCCTCTAT 100 743 1047473 2369 2388 12658 12677 CAACCCCTACTTGTATGCCT  73 744 1047489 2429 2448 12718 12737 AGAGGATGAGTCACTTCCTT 116 745 1047505 2469 2488 12758 12777 CATGGATACATCCCCTTTCT  83 746 1047521 2748 2767 13037 13056 CAGTGTCTTCACTTTGCTCG  69 747 1047537 2846 2865 13135 13154 AGTCTGCTCACCAGTCTGCT 103 748 1047553 2884 2903 13173 13192 GGGCCCTCCCAGTCCCATCT 105 749 1047569 2957 2976 13246 13265 GAAAGGACACCAAGTCTTGG 117 750 1047585 3050 3069 13339 13358 TTTGTCTTTATTTTTCCTCA  20 751 1047601 N/A N/A  8751  8770 GATTTTCCCCGTCTTTGGTG  38 752 1047617 N/A N/A  8901  8920 GTGAGGCTCACTCCCTGTCA  95 753 1047633 N/A N/A  9060  9079 ACCACCTTTACCACTAACAA 108 754 1047649 N/A N/A  9107  9126 GCTTGCTCAGAGGCCCCAGA  59 755 1047665 N/A N/A  9315  9334 TAATATTTAACATTAAGAGC 135 756 1047681 N/A N/A  8406  8425 GACTCCAGTGCCCTTCCCAC 109 757 1047697 N/A N/A  8473  8492 TGGATCCCTTTGCCCTGATC  90 758 1047713 N/A N/A  8557  8576 GCTGCAAGCCCCACCTAGAA  69 759 1047729 N/A N/A  8692  8711 AACAGAAAACACTCAGAAGG  97 760 1047745 N/A N/A  3985  4004 AAGGCCCCCCTTCCCCATCC 109 761 1047761 N/A N/A  4158  4177 TGCGGGCATCAGATCCCCGG 138 762 1047777 N/A N/A  4313  4332 TCCTCCTTTATATGGACACA 111 763 1047793 N/A N/A  4341  4360 ATGGACATCAACCTTCTCCG  74 764 1047809 N/A N/A  4378  4397 TCCCTCACTTTCTTCACCCC 100 765 1047825 N/A N/A  4514  4533 CCCAGGTAACCACCTTTTGA  92 766 1047841 N/A N/A  4689  4708 AAGGTGCCTTATCAGGGTTG  74 767 1047857 N/A N/A  4716  4735 TGTGGGCTTTTGCCTTAACT 103 768 1047873 N/A N/A  4841  4860 TACCTGCCTCAGTCTCCCTT 130 769 1047889 N/A N/A  4998  5017 AATCCTTTCCTCCCTCCCCT 129 770 1047905 N/A N/A  5103  5122 TCCCCATTCCTCAGCCTTGC 112 771 1047921 N/A N/A  5317  5336 TGTCTCTCCCTCTCTCAGTT  88 772 1047937 N/A N/A  5438  5457 CTTGTCTCTTTCCGTCTCCC 105 773 1047953 N/A N/A  5739  5758 GCAGGCTGGCCCACAGGCAG  83 774 1047969 N/A N/A  5816  5835 AGAGGATATTCTCCCAGCTT  80 775 1047985 N/A N/A  5994  6013 GCACCTACTTCATAGTAAGG  65 776 1048001 N/A N/A  6089  6108 AGTTGGAAATCCAACTCTAC  88 777 1048017 N/A N/A  6157  6176 AGAGGCTCAGTAACCCAAAA  90 778 1048033 N/A N/A  6306  6325 CCCCTCTACAGTGTCTTTCC 101 779 1048049 N/A N/A  6364  6383 TGGCCCTCCCTTCTTCTCTT  89 780 1048065 N/A N/A  6497  6516 GGCCCTCACCCTGCTCAGAC 143 781 1048081 N/A N/A  6571  6590 CCTCGCCTGGCCTCACCCTG 116 782 1048097 N/A N/A  7056  7075 ACTGTGACCCATGGATGCGG  98 783 1048113 N/A N/A  7343  7362 CGCCCGTCCCCGTCCTGCCC  92 784 1048129 N/A N/A  7835  7854 TGAAAAAGACTCAGTCCCTG 103 785 1048145 N/A N/A  7947  7966 ATTGGTTGAATCCATCCATC 106 786 1048161 N/A N/A  8199  8218 TCCTAGCTTTTTCCCCAGCA  92 787 1048177 N/A N/A  9318  9337 CACTAATATTTAACATTAAG 120 788 1048193 N/A N/A  9475  9494 GACATGCATATCTAGTGGTC  64 789 1048209 N/A N/A  9595  9614 CTTTCCTACTTCTCTCTGCC 107 790 1048225 N/A N/A  9634  9653 TGCTCAATACACATAGGTCC  95 791 1048241 N/A N/A  9806  9825 GAAGTCCCAACTTAGCCACC 101 792 1048257 N/A N/A 10579 10598 CCTGGCTTTCTGAAAACCCA  70 793 1048273 N/A N/A 10683 10702 GGCTGGAGAGCCCCCAAATC 144 794 1048289 N/A N/A 10766 10785 GGCTTCCCCATCGCACCCCC 117 795 1048305 N/A N/A 10930 10949 ATCCCTCCTTACCTCTCCAT 106 796 1048321 N/A N/A 10998 11017 CCCCAGGCTTTCCTCCATGG  81 797 1048337 N/A N/A 11113 11132 CCTTAGAACAGCCTATGGAG 162 798 1048353 N/A N/A 11305 11324 TGGTAGTTTCCTTTTACCAA  89 799 1048369 N/A N/A 11339 11358 GGCGGATACGCCAAATCCCC  93 800 1048385 N/A N/A 11547 11566 ACAGACCCCAGGTCCACCAC 104 801

TABLE 12 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047154  120  139  3568  3587 GGCCCCCCACCATCATCTCC  97 802 1047170  226  245  3674  3693 GCATTGAGTGCCCCAGCCAG 128 803 1047186  471  490  3919  3938 CCCTCTCAACCTCCAGCCGG   3* 804 1047202  541  560  4926  4945 GCTTCCAGCCTCAGGTTGGT  18* 805 1047218  628  647  5217  5236 TCCAGCGACTCAATCTTCCT 100 806 1047234  723  742  5609  5628 CGTCAAGCTCCACATGGACC 144 807 1047250 1017 1036  7589  7608 AACTGGCCGCCTCCCGCACG 127 808 1047266 1206 1225  8355  8374 GGTTGGAGAAGGTCTGCACG  77 809 1047282 1275 1294 10889 10908 CCACGATGTTCCTCTTGAGG 157 810 1047298 1322 1341 11611 11630 CTTGGACTCCTTAATGACCT  64 811 1047314 1395 1414 11684 11703 GCTCGGGCCCCTCATGAGAC 100 812 1047330 1606 1625 11895 11914 CATGCCCCTCCAGACTGCCC 106 813 1047346 1657 1676 11946 11965 CGCCCTCCTCCCCTTCTCTC  79 814 1047362 1718 1737 12007 12026 GGCAACAGTTTCCATAACAA  31 815 1047378 1771 1790 12060 12079 TGAGGAAACTCAAAGGCACA  79 816 1047394 1835 1854 12124 12143 CAGGGCTACCTTGTCTGTGG  40 817 1047410 2025 2044 12314 12333 ATCTTAGACTGATCAGGGTC  67 818 1047426 2167 2186 12456 12475 AGCTCCCACCTGCCCACAGC  94 819 1047442 2226 2245 12515 12534 CCTCCCAGCACCTCATCCCT  97 820 1047458 2268 2287 12557 12576 TCTCTGTACCCACAGCTGGG  84 821 1047474 2371 2390 12660 12679 CACAACCCCTACTTGTATGC  61 822 1047490 2442 2461 12731 12750 TTCAGCATCTTCAAGAGGAT  92 823 1047506 2470 2489 12759 12778 CCATGGATACATCCCCTTTC 151 824 1047522 2749 2768 13038 13057 CCAGTGTCTTCACTTTGCTC  44 825 1047538 2847 2866 13136 13155 CAGTCTGCTCACCAGTCTGC 103 826 1047554 2885 2904 13174 13193 TGGGCCCTCCCAGTCCCATC 172 827 1047570 2958 2977 13247 13266 GGAAAGGACACCAAGTCTTG  90 828 1047586 3051 3070 13340 13359 ATTTGTCTTTATTTTTCCTC  32 829 1047602 N/A N/A  8754  8773 TGTGATTTTCCCCGTCTTTG  54 830 1047618 N/A N/A  8902  8921 GGTGAGGCTCACTCCCTGTC  65 831 1047634 N/A N/A  9061  9080 CACCACCTTTACCACTAACA  88 832 1047650 N/A N/A  9109  9128 CTGCTTGCTCAGAGGCCCCA 115 833 1047666 N/A N/A  8372  8391 CTGACCTCGAATCTGCAGGT  84 834 1047682 N/A N/A  8412  8431 GGGCAGGACTCCAGTGCCCT 118 835 1047698 N/A N/A  8474  8493 CTGGATCCCTTTGCCCTGAT 100 836 1047714 N/A N/A  8626  8645 CACCCAGTTCTGCTGTCGAA  94 837 1047730 N/A N/A  8696  8715 CAAAAACAGAAAACACTCAG 160 838 1047746 N/A N/A  3987  4006 ACAAGGCCCCCCTTCCCCAT  57 839 1047762 N/A N/A  4215  4234 CACTGCTTTCCCCAGTAGGG  48 840 1047778 N/A N/A  4314  4333 CTCCTCCTTTATATGGACAC  96 841 1047794 N/A N/A  4345  4364 CCTCATGGACATCAACCTTC  92 842 1047810 N/A N/A  4379  4398 TTCCCTCACTTTCTTCACCC 102 843 1047826 N/A N/A  4515  4534 TCCCAGGTAACCACCTTTTG  76 844 1047842 N/A N/A  4691  4710 CTAAGGTGCCTTATCAGGGT  78 845 1047858 N/A N/A  4794  4813 ACCCAGGACCAGTAGAGCAG  84 846 1047874 N/A N/A  4843  4862 AATACCTGCCTCAGTCTCCC 106 847 1047890 N/A N/A  4999  5018 CAATCCTTTCCTCCCTCCCC 109 848 1047906 N/A N/A  5104  5123 CTCCCCATTCCTCAGCCTTG  90 849 1047922 N/A N/A  5334  5353 CTCAGCTTCTCTGTCTCTGT 123 850 1047938 N/A N/A  5465  5484 CCCCTCGGCCAGGAGTTCGA 123 851 1047954 N/A N/A  5781  5800 CCCCATTCTCTTGTACAGAG  98 852 1047970 N/A N/A  5817  5836 GAGAGGATATTCTCCCAGCT 151 853 1047986 N/A N/A  6001  6020 AAGAACAGCACCTACTTCAT  85 854 1048002 N/A N/A  6090  6109 GAGTTGGAAATCCAACTCTA  97 855 1048018 N/A N/A  6159  6178 GTAGAGGCTCAGTAACCCAA  51 856 1048034 N/A N/A  6310  6329 ATGCCCCCTCTACAGTGTCT  71 857 1048050 N/A N/A  6366  6385 AATGGCCCTCCCTTCTTCTC 177 858 1048066 N/A N/A  6503  6522 CCATCGGGCCCTCACCCTGC 110 859 1048082 N/A N/A  6951  6970 ACCTTCCACACTGACAGCTG 169 860 1048098 N/A N/A  7058  7077 CAACTGTGACCCATGGATGC  78 861 1048114 N/A N/A  7349  7368 CTGCTCCGCCCGTCCCCGTC 120 862 1048130 N/A N/A  7836  7855 CTGAAAAAGACTCAGTCCCT 118 863 1048146 N/A N/A  7949  7968 ATATTGGTTGAATCCATCCA 102 864 1048162 N/A N/A  8211  8230 TCTAACTCCATCTCCTAGCT  98 865 1048178 N/A N/A  9319  9338 TCACTAATATTTAACATTAA  84 866 1048194 N/A N/A  9492  9511 GCTGAATTAAGTCCTGAGAC  55 867 1048210 N/A N/A  9596  9615 TCTTTCCTACTTCTCTCTGC  82 868 1048226 N/A N/A  9662  9681 AGGCTGTTAAACATGTGGCA  71 869 1048242 N/A N/A  9807  9826 AGAAGTCCCAACTTAGCCAC  85 870 1048258 N/A N/A 10580 10599 ACCTGGCTTTCTGAAAACCC  79 871 1048274 N/A N/A 10698 10717 AGGCTCTTCCAAACGGGCTG 103 872 1048290 N/A N/A 10768 10787 CCGGCTTCCCCATCGCACCC  81 873 1048306 N/A N/A 10931 10950 AATCCCTCCTTACCTCTCCA 116 874 1048322 N/A N/A 11004 11023 TGCCAGCCCCAGGCTTTCCT  66 875 1048338 N/A N/A 11117 11136 CTCCCCTTAGAACAGCCTAT  88 876 1048354 N/A N/A 11306 11325 CTGGTAGTTTCCTTTTACCA 113 877 1048370 N/A N/A 11340 11359 TGGCGGATACGCCAAATCCC  75 878 1048386 N/A N/A 11555 11574 GAGTTCACACAGACCCCAGG  85 879

TABLE 13 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047155  121  140  3569  3588 AGGCCCCCCACCATCATCTC 124 880 1047171  228  247  3676  3695 CAGCATTGAGTGCCCCAGCC 104 881 1047187  472  491  3920  3939 TCCCTCTCAACCTCCAGCCG   7* 882 1047203  542  561  4927  4946 GGCTTCCAGCCTCAGGTTGG  18* 883 1047219  629  648  5218  5237 CTCCAGCGACTCAATCTTCC  86 884 1047235  761  780  5647  5666 GATCTCTTTCAGGGCTGCGG  51 885 1047251 1019 1038  7591  7610 ATAACTGGCCGCCTCCCGCA 143 886 1047267 1223 1242 N/A N/A GGTTTCTCGAATCTGCAGGT  65 887 1047283 1280 1299 10894 10913 CTTCACCACGATGTTCCTCT  73 888 1047299 1323 1342 11612 11631 GCTTGGACTCCTTAATGACC  91 889 1047315 1396 1415 11685 11704 TGCTCGGGCCCCTCATGAGA  84 890 1047331 1607 1626 11896 11915 CCATGCCCCTCCAGACTGCC  66 891 1047347 1658 1677 11947 11966 CCGCCCTCCTCCCCTTCTCT  73 892 1047363 1721 1740 12010 12029 TCTGGCAACAGTTTCCATAA  66 893 1047379 1772 1791 12061 12080 CTGAGGAAACTCAAAGGCAC 100 894 1047395 1837 1856 12126 12145 GCCAGGGCTACCTTGTCTGT  80 895 1047411 2026 2045 12315 12334 CATCTTAGACTGATCAGGGT  72 896 1047427 2169 2188 12458 12477 CAAGCTCCCACCTGCCCACA 111 897 1047443 2232 2251 12521 12540 TCTATCCCTCCCAGCACCTC  80 898 1047459 2269 2288 12558 12577 CTCTCTGTACCCACAGCTGG  85 899 1047475 2372 2391 12661 12680 CCACAACCCCTACTTGTATG  69 900 1047491 2443 2462 12732 12751 TTTCAGCATCTTCAAGAGGA 121 901 1047507 2471 2490 12760 12779 CCCATGGATACATCCCCTTT  79 902 1047523 2751 2770 13040 13059 AGCCAGTGTCTTCACTTTGC  60 903 1047539 2855 2874 13144 13163 GATCCCACCAGTCTGCTCAC  84 904 1047555 2886 2905 13175 13194 GTGGGCCCTCCCAGTCCCAT  81 905 1047571 3010 3029 13299 13318 TGCCCTGAAGATTAGCAGCA 103 906 1047587 3052 3071 13341 13360 CATTTGTCTTTATTTTTCCT  14 907 1047603 N/A N/A  8755  8774 TTGTGATTTTCCCCGTCTTT  75 908 1047619 N/A N/A  8969  8988 ACGCAGTCCAGGCCCTTTAG  57 909 1047635 N/A N/A  9063  9082 CTCACCACCTTTACCACTAA 113 910 1047651 N/A N/A  9128  9147 AGAGGTGAGACAGAGGCTGC 112 911 1047667 N/A N/A  8374  8393 TACTGACCTCGAATCTGCAG  85 912 1047683 N/A N/A  8432  8451 CCTACAGGCCCTGGAGGAGG  85 913 1047699 N/A N/A  8476  8495 AGCTGGATCCCTTTGCCCTG  97 914 1047715 N/A N/A  8630  8649 CTCTCACCCAGTTCTGCTGT  77 915 1047731 N/A N/A  8723  8742 CCCTGTAGTGACAAGCAGTT  84 916 1047747 N/A N/A  3997  4016 CCTTCTGCTCACAAGGCCCC  93 917 1047763 N/A N/A  4257  4276 AAGCCCAGCCATGAATGAAA  83 918 1047779 N/A N/A  4316  4335 AACTCCTCCTTTATATGGAC  91 919 1047795 N/A N/A  4353  4372 CCAATCTCCCTCATGGACAT  66 920 1047811 N/A N/A  4386  4405 CTGCTCTTTCCCTCACTTTC 103 921 1047827 N/A N/A  4516  4535 ATCCCAGGTAACCACCTTTT  85 922 1047843 N/A N/A  4692  4711 ACTAAGGTGCCTTATCAGGG 117 923 1047859 N/A N/A  4795  4814 CACCCAGGACCAGTAGAGCA  56 924 1047875 N/A N/A  4848  4867 ACTTGAATACCTGCCTCAGT  85 925 1047891 N/A N/A  5001  5020 ATCAATCCTTTCCTCCCTCC 102 926 1047907 N/A N/A  5107  5126 CTTCTCCCCATTCCTCAGCC  56 927 1047923 N/A N/A  5349  5368 AGTGTCTCTCTCAGTCTCAG  83 928 1047939 N/A N/A  5477  5496 CTCTTCTGCCTGCCCCTCGG 101 929 1047955 N/A N/A  5783  5802 TCCCCCATTCTCTTGTACAG  49 930 1047971 N/A N/A  5818  5837 GGAGAGGATATTCTCCCAGC  93 931 1047987 N/A N/A  6007  6026 CTGTCAAAGAACAGCACCTA 112 932 1048003 N/A N/A  6091  6110 AGAGTTGGAAATCCAACTCT  92 933 1048019 N/A N/A  6201  6220 GGTCAGACACCTCTCTGTGT  82 934 1048035 N/A N/A  6321  6340 GCCTAGCCCAAATGCCCCCT  71 935 1048051 N/A N/A  6384  6403 GCTCTGTCCTCCACTAGGAA  97 936 1048067 N/A N/A  6504  6523 CCCATCGGGCCCTCACCCTG  88 937 1048083 N/A N/A  6954  6973 AGAACCTTCCACACTGACAG  76 938 1048099 N/A N/A  7060  7079 AACAACTGTGACCCATGGAT  74 939 1048115 N/A N/A  7351  7370 CCCTGCTCCGCCCGTCCCCG  98 940 1048131 N/A N/A  7837  7856 GCTGAAAAAGACTCAGTCCC  84 941 1048147 N/A N/A  7973  7992 GTCCTTGGCCTTGAGGCCTA  95 942 1048163 N/A N/A  8213  8232 AGTCTAACTCCATCTCCTAG  88 943 1048179 N/A N/A  9322  9341 CATTCACTAATATTTAACAT 110 944 1048195 N/A N/A  9507  9526 GTTAGCCTTTCTGATGCTGA  53 945 1048211 N/A N/A  9597  9616 CTCTTTCCTACTTCTCTCTG  91 946 1048227 N/A N/A  9708  9727 GGCCTACTTCTCTAGGTGGG  82 947 1048243 N/A N/A  9808  9827 AAGAAGTCCCAACTTAGCCA 141 948 1048259 N/A N/A 10602 10621 CCTCTGCAAGCCCTGGCCTG  95 949 1048275 N/A N/A 10699 10718 TAGGCTCTTCCAAACGGGCT  89 950 1048291 N/A N/A 10769 10788 CCCGGCTTCCCCATCGCACC  79 951 1048307 N/A N/A 10934 10953 CCAAATCCCTCCTTACCTCT 156 952 1048323 N/A N/A 11017 11036 GGAACCTTCTATGTGCCAGC  74 953 1048339 N/A N/A 11132 11151 GCTTTGGTACCAAGGCTCCC 192 954 1048355 N/A N/A 11307 11326 CCTGGTAGTTTCCTTTTACC  52 955 1048371 N/A N/A 11379 11398 GCTGGAGTAAGATGAGCTCC  86 956 1048387 N/A N/A 11570 11589 CAGTGCAACAGTTAGGAGTT  66 957

TABLE 14 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047156  122  141  3570  3589 CAGGCCCCCCACCATCATCT  90  958 1047172  237  256  3685  3704 CCTTGAAGCCAGCATTGAGT  89  959 1047188  473  492  3921  3940 GTCCCTCTCAACCTCCAGCC   6*  960 1047204  545  564  4930  4949 CTCGGCTTCCAGCCTCAGGT  37*  961 1047220  634  653  5223  5242 TCCTCCTCCAGCGACTCAAT  94  962 1047236  766  785  5652  5671 GTGCGGATCTCTTTCAGGGC  63  963 1047252 1020 1039  7592  7611 GATAACTGGCCGCCTCCCGC 113  964 1047268 1225 1244 N/A N/A CTGGTTTCTCGAATCTGCAG  83  965 1047284 1283 1302 10897 10916 GGTCTTCACCACGATGTTCC  94  966 1047300 1345 1364 11634 11653 CACATCACATCCTTGTGCTC  75  967 1047316 1398 1417 11687 11706 TCTGCTCGGGCCCCTCATGA  96  968 1047332 1608 1627 11897 11916 GCCATGCCCCTCCAGACTGC  73  969 1047348 1659 1678 11948 11967 CCCGCCCTCCTCCCCTTCTC 107  970 1047364 1723 1742 12012 12031 TCTCTGGCAACAGTTTCCAT  65  971 1047380 1773 1792 12062 12081 CCTGAGGAAACTCAAAGGCA  92  972 1047396 1838 1857 12127 12146 GGCCAGGGCTACCTTGTCTG  67  973 1047412 2027 2046 12316 12335 CCATCTTAGACTGATCAGGG  76  974 1047428 2170 2189 12459 12478 TCAAGCTCCCACCTGCCCAC 127  975 1047444 2233 2252 12522 12541 CTCTATCCCTCCCAGCACCT  42  976 1047460 2270 2289 12559 12578 CCTCTCTGTACCCACAGCTG  94  977 1047476 2373 2392 12662 12681 CCCACAACCCCTACTTGTAT 115  978 1047492 2449 2468 12738 12757 CTCCTGTTTCAGCATCTTCA  45  979 1047508 2472 2491 12761 12780 CCCCATGGATACATCCCCTT  86  980 1047524 2759 2778 13048 13067 GGAATATGAGCCAGTGTCTT  63  981 1047540 2857 2876 13146 13165 CAGATCCCACCAGTCTGCTC  89  982 1047556 2889 2908 13178 13197 GAAGTGGGCCCTCCCAGTCC 113  983 1047572 3011 3030 13300 13319 GTGCCCTGAAGATTAGCAGC  85  984 1047588 3053 3072 13342 13361 GCATTTGTCTTTATTTTTCC  11  985 1047604 N/A N/A  8756  8775 CTTGTGATTTTCCCCGTCTT  74  986 1047620 N/A N/A  8972  8991 ATGACGCAGTCCAGGCCCTT  78  987 1047636 N/A N/A  9064  9083 ACTCACCACCTTTACCACTA  85  988 1047652 N/A N/A  9129  9148 AAGAGGTGAGACAGAGGCTG  66  989 1047668 N/A N/A  8375  8394 GTACTGACCTCGAATCTGCA  96  990 1047684 N/A N/A  8437  8456 AGCAACCTACAGGCCCTGGA 116  991 1047700 N/A N/A  8477  8496 GAGCTGGATCCCTTTGCCCT  82  992 1047716 N/A N/A  8631  8650 GCTCTCACCCAGTTCTGCTG  83  993 1047732 N/A N/A  8725  8744 CCCCCTGTAGTGACAAGCAG  71  994 1047748 N/A N/A  4051  4070 GAGGTTCGGCCCCTCCCTGA  68  995 1047764 N/A N/A  4258  4277 AAAGCCCAGCCATGAATGAA 111  996 1047780 N/A N/A  4317  4336 CAACTCCTCCTTTATATGGA 104  997 1047796 N/A N/A  4355  4374 ATCCAATCTCCCTCATGGAC  78  998 1047812 N/A N/A  4388  4407 GCCTGCTCTTTCCCTCACTT  98  999 1047828 N/A N/A  4520  4539 TCTGATCCCAGGTAACCACC  81 1000 1047844 N/A N/A  4693  4712 TACTAAGGTGCCTTATCAGG  92 1001 1047860 N/A N/A  4800  4819 ACCCCCACCCAGGACCAGTA  94 1002 1047876 N/A N/A  4851  4870 GACACTTGAATACCTGCCTC  89 1003 1047892 N/A N/A  5002  5021 CATCAATCCTTTCCTCCCTC  79 1004 1047908 N/A N/A  5109  5128 TCCTTCTCCCCATTCCTCAG  88 1005 1047924 N/A N/A  5355  5374 TCTCTGAGTGTCTCTCTCAG  92 1006 1047940 N/A N/A  5478  5497 CCTCTTCTGCCTGCCCCTCG 111 1007 1047956 N/A N/A  5784  5803 TTCCCCCATTCTCTTGTACA  98 1008 1047972 N/A N/A  5836  5855 GGTGAAAGTCAGTCACCTGG  90 1009 1047988 N/A N/A  6009  6028 ATCTGTCAAAGAACAGCACC  96 1010 1048004 N/A N/A  6092  6111 TAGAGTTGGAAATCCAACTC  92 1011 1048020 N/A N/A  6211  6230 ACACCTTCCAGGTCAGACAC  71 1012 1048036 N/A N/A  6322  6341 TGCCTAGCCCAAATGCCCCC 111 1013 1048052 N/A N/A  6386  6405 AGGCTCTGTCCTCCACTAGG  87 1014 1048068 N/A N/A  6513  6532 CCTCCCAGCCCCATCGGGCC 146 1015 1048084 N/A N/A  6955  6974 CAGAACCTTCCACACTGACA 123 1016 1048100 N/A N/A  7065  7084 TTCCCAACAACTGTGACCCA  69 1017 1048116 N/A N/A  7387  7406 TGGCCCTTCTCCCCTGGCAT 122 1018 1048132 N/A N/A  7840  7859 AAGGCTGAAAAAGACTCAGT  82 1019 1048148 N/A N/A  7982  8001 GTGACCCAAGTCCTTGGCCT  85 1020 1048164 N/A N/A  8217  8236 GGAAAGTCTAACTCCATCTC  93 1021 1048180 N/A N/A  9323  9342 ACATTCACTAATATTTAACA  91 1022 1048196 N/A N/A  9510  9529 CTGGTTAGCCTTTCTGATGC  51 1023 1048212 N/A N/A  9599  9618 TCCTCTTTCCTACTTCTCTC  73 1024 1048228 N/A N/A  9709  9728 GGGCCTACTTCTCTAGGTGG  48 1025 1048244 N/A N/A  9813  9832 AGCTCAAGAAGTCCCAACTT  87 1026 1048260 N/A N/A 10616 10635 GCTTCATTTCAGCCCCTCTG  82 1027 1048276 N/A N/A 10700 10719 CTAGGCTCTTCCAAACGGGC  94 1028 1048292 N/A N/A 10771 10790 TGCCCGGCTTCCCCATCGCA  98 1029 1048308 N/A N/A 10936 10955 GCCCAAATCCCTCCTTACCT  95 1030 1048324 N/A N/A 11021 11040 GCTGGGAACCTTCTATGTGC  64 1031 1048340 N/A N/A 11133 11152 GGCTTTGGTACCAAGGCTCC  89 1032 1048356 N/A N/A 11309 11328 CCCCTGGTAGTTTCCTTTTA  94 1033 1048372 N/A N/A 11433 11452 TGGTGAGATAACACTGGGAA  51 1034 1048388 N/A N/A 11571 11590 ACAGTGCAACAGTTAGGAGT  73 1035

TABLE 15 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047157  123  142  3571  3590 CCAGGCCCCCCACCATCATC 116 1036 1047173  239  258  3687  3706 CTCCTTGAAGCCAGCATTGA  95 1037 1047189  474  493  3922  3941 TGTCCCTCTCAACCTCCAGC  11* 1038 1047205  547  566  4932  4951 TTCTCGGCTTCCAGCCTCAG  31* 1039 1047221  637  656  5226  5245 ATCTCCTCCTCCAGCGACTC  83 1040 1047237  786  805  5672  5691 ACGCCATTGCCTCATACTGC 104 1041 1047253 1021 1040  7593  7612 TGATAACTGGCCGCCTCCCG  94 1042 1047269 1227 1246 N/A N/A GGCTGGTTTCTCGAATCTGC  92 1043 1047285 1285 1304 10899 10918 ACGGTCTTCACCACGATGTT  86 1044 1047301 1346 1365 11635 11654 TCACATCACATCCTTGTGCT  79 1045 1047317 1406 1425 11695 11714 ATCCTGCTTCTGCTCGGGCC  98 1046 1047333 1610 1629 11899 11918 TGGCCATGCCCCTCCAGACT  74 1047 1047349 1660 1679 11949 11968 CCCCGCCCTCCTCCCCTTCT  98 1048 1047365 1724 1743 12013 12032 ATCTCTGGCAACAGTTTCCA  60 1049 1047381 1774 1793 12063 12082 GCCTGAGGAAACTCAAAGGC  91 1050 1047397 1839 1858 12128 12147 TGGCCAGGGCTACCTTGTCT  82 1051 1047413 2033 2052 12322 12341 CCCCACCCATCTTAGACTGA  84 1052 1047429 2172 2191 12461 12480 AATCAAGCTCCCACCTGCCC  45 1053 1047445 2234 2253 12523 12542 CCTCTATCCCTCCCAGCACC 104 1054 1047461 2275 2294 12564 12583 CTTGACCTCTCTGTACCCAC  67 1055 1047477 2375 2394 12664 12683 CACCCACAACCCCTACTTGT  97 1056 1047493 2450 2469 12739 12758 TCTCCTGTTTCAGCATCTTC  58 1057 1047509 2478 2497 12767 12786 CCCTGCCCCCATGGATACAT  76 1058 1047525 2765 2784 13054 13073 GCTGCAGGAATATGAGCCAG  84 1059 1047541 2858 2877 13147 13166 ACAGATCCCACCAGTCTGCT  98 1060 1047557 2890 2909 13179 13198 TGAAGTGGGCCCTCCCAGTC  87 1061 1047573 3016 3035 13305 13324 CAGCAGTGCCCTGAAGATTA  45 1062 1047589 3054 3073 13343 13362 AGCATTTGTCTTTATTTTTC  17 1063 1047605 N/A N/A  8757  8776 CCTTGTGATTTTCCCCGTCT  64 1064 1047621 N/A N/A  9023  9042 TACAGTTACTCTGTACCACG 118 1065 1047637 N/A N/A  9065  9084 GACTCACCACCTTTACCACT  87 1066 1047653 N/A N/A  9199  9218 GATGAAAGAATAAAGCAGAG 102 1067 1047669 N/A N/A  8376  8395 TGTACTGACCTCGAATCTGC  90 1068 1047685 N/A N/A  8439  8458 GGAGCAACCTACAGGCCCTG 104 1069 1047701 N/A N/A  8478  8497 AGAGCTGGATCCCTTTGCCC  87 1070 1047717 N/A N/A  8632  8651 AGCTCTCACCCAGTTCTGCT  62 1071 1047733 N/A N/A  8731  8750 CTTTTGCCCCCTGTAGTGAC  56 1072 1047749 N/A N/A  4088  4107 GTGCCCCATCAAGAGGTAGG 147 1073 1047765 N/A N/A  4260  4279 ACAAAGCCCAGCCATGAATG 142 1074 1047781 N/A N/A  4318  4337 CCAACTCCTCCTTTATATGG  74 1075 1047797 N/A N/A  4356  4375 AATCCAATCTCCCTCATGGA  93 1076 1047813 N/A N/A  4390  4409 CTGCCTGCTCTTTCCCTCAC  99 1077 1047829 N/A N/A  4521  4540 CTCTGATCCCAGGTAACCAC  96 1078 1047845 N/A N/A  4700  4719 AACTCATTACTAAGGTGCCT 101 1079 1047861 N/A N/A  4801  4820 CACCCCCACCCAGGACCAGT  90 1080 1047877 N/A N/A  4852  4871 GGACACTTGAATACCTGCCT  84 1081 1047893 N/A N/A  5003  5022 CCATCAATCCTTTCCTCCCT  82 1082 1047909 N/A N/A  5112  5131 GGCTCCTTCTCCCCATTCCT  92 1083 1047925 N/A N/A  5368  5387 TGTTTCTCTCCTCTCTCTGA 113 1084 1047941 N/A N/A  5484  5503 TTGTGTCCTCTTCTGCCTGC  93 1085 1047957 N/A N/A  5786  5805 CCTTCCCCCATTCTCTTGTA  87 1086 1047973 N/A N/A  5842  5861 TTCTCTGGTGAAAGTCAGTC  95 1087 1047989 N/A N/A  6012  6031 CTCATCTGTCAAAGAACAGC 102 1088 1048005 N/A N/A  6110  6129 CTCCCAAGTGAGATGTGCTA 112 1089 1048021 N/A N/A  6212  6231 CACACCTTCCAGGTCAGACA  70 1090 1048037 N/A N/A  6323  6342 CTGCCTAGCCCAAATGCCCC  86 1091 1048053 N/A N/A  6396  6415 TTCTGCCTCCAGGCTCTGTC 117 1092 1048069 N/A N/A  6519  6538 GGAGGTCCTCCCAGCCCCAT  94 1093 1048085 N/A N/A  6956  6975 CCAGAACCTTCCACACTGAC  79 1094 1048101 N/A N/A  7069  7088 ACTTTTCCCAACAACTGTGA  84 1095 1048117 N/A N/A  7388  7407 CTGGCCCTTCTCCCCTGGCA  81 1096 1048133 N/A N/A  7841  7860 CAAGGCTGAAAAAGACTCAG 121 1097 1048149 N/A N/A  7985  8004 AGGGTGACCCAAGTCCTTGG  89 1098 1048165 N/A N/A  8219  8238 CAGGAAAGTCTAACTCCATC  80 1099 1048181 N/A N/A  9324  9343 CACATTCACTAATATTTAAC  93   21 1048197 N/A N/A  9512  9531 GCCTGGTTAGCCTTTCTGAT  59 1100 1048213 N/A N/A  9604  9623 CCCTTTCCTCTTTCCTACTT  71 1101 1048229 N/A N/A  9727  9746 TGTAAAATAAGGATGATGGG 101 1102 1048245 N/A N/A  9816  9835 CAGAGCTCAAGAAGTCCCAA 101 1103 1048261 N/A N/A 10617 10636 GGCTTCATTTCAGCCCCTCT  73 1104 1048277 N/A N/A 10702 10721 GCCTAGGCTCTTCCAAACGG 105 1105 1048293 N/A N/A 10784 10803 CCATCCTCTCCCATGCCCGG 100 1106 1048309 N/A N/A 10937 10956 GGCCCAAATCCCTCCTTACC 118 1107 1048325 N/A N/A 11058 11077 CTCTCCTCCAGAATTCCCTG  76 1108 1048341 N/A N/A 11149 11168 TAGGATCCCATCTAGTGGCT  51 1109 1048357 N/A N/A 11316 11335 TCCCATTCCCCTGGTAGTTT  57 1110 1048373 N/A N/A 11435 11454 GGTGGTGAGATAACACTGGG  88 1111 1048389 N/A N/A 11608 11627 GGACTCCTTAATGACCTGCA  53 1112

TABLE 16 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047158  124  143  3572  3591 GCCAGGCCCCCCACCATCAT 102 1113 1047174  240  259  3688  3707 TCTCCTTGAAGCCAGCATTG 109 1114 1047190  477  496  3925  3944 GATTGTCCCTCTCAACCTCC  17* 1115 1047206  563  582  4948  4967 ATAGGCAGCCAGGTTGTTCT  35* 1116 1047222  638  657  5227  5246 GATCTCCTCCTCCAGCGACT  75 1117 1047238  809  828  5695  5714 TTCGGCTTCATGCATGTTGC 123 1118 1047254 1026 1045  7598  7617 CCTCCTGATAACTGGCCGCC 117 1119 1047270 1228 1247 N/A N/A AGGCTGGTTTCTCGAATCTG 117 1120 1047286 1286 1305 10900 10919 CACGGTCTTCACCACGATGT  89 1121 1047302 1348 1367 11637 11656 CCTCACATCACATCCTTGTG 103 1122 1047318 1413 1432 11702 11721 AGCAACTATCCTGCTTCTGC  58 1123 1047334 1612 1631 11901 11920 GCTGGCCATGCCCCTCCAGA 114 1124 1047350 1661 1680 11950 11969 CCCCCGCCCTCCTCCCCTTC  90 1125 1047366 1731 1750 12020 12039 AACCTCCATCTCTGGCAACA  59 1126 1047382 1782 1801 12071 12090 CTCCAGCAGCCTGAGGAAAC 131 1127 1047398 1844 1863 12133 12152 GCCTCTGGCCAGGGCTACCT 100 1128 1047414 2034 2053 12323 12342 TCCCCACCCATCTTAGACTG 113 1129 1047430 2178 2197 12467 12486 GCTGAGAATCAAGCTCCCAC  54 1130 1047446 2235 2254 12524 12543 CCCTCTATCCCTCCCAGCAC 100 1131 1047462 2278 2297 12567 12586 GGGCTTGACCTCTCTGTACC  73 1132 1047478 2376 2395 12665 12684 TCACCCACAACCCCTACTTG  81 1133 1047494 2451 2470 12740 12759 CTCTCCTGTTTCAGCATCTT  61 1134 1047510 2481 2500 12770 12789 ATGCCCTGCCCCCATGGATA  83 1135 1047526 2777 2796 13066 13085 CCCGGCCTCCAGGCTGCAGG  89 1136 1047542 2859 2878 13148 13167 CACAGATCCCACCAGTCTGC  85 1137 1047558 2901 2920 13190 13209 GAGGAGAACCCTGAAGTGGG  95 1138 1047574 3018 3037 13307 13326 AGCAGCAGTGCCCTGAAGAT  78 1139 1047590 3055 3074 13344 13363 CAGCATTTGTCTTTATTTTT  21 1140 1047606 N/A N/A  8758  8777 ACCTTGTGATTTTCCCCGTC  70 1141 1047622 N/A N/A  9024  9043 GTACAGTTACTCTGTACCAC 138 1142 1047638 N/A N/A  9066  9085 GGACTCACCACCTTTACCAC  68 1143 1047654 N/A N/A  9200  9219 GGATGAAAGAATAAAGCAGA  85 1144 1047670 N/A N/A  8377  8396 CTGTACTGACCTCGAATCTG  93 1145 1047686 N/A N/A  8443  8462 GTCTGGAGCAACCTACAGGC  79 1146 1047702 N/A N/A  8495  8514 CACGAAGGCCCCCAGGGAGA  77 1147 1047718 N/A N/A  8633  8652 AAGCTCTCACCCAGTTCTGC 108 1148 1047734 N/A N/A  8733  8752 TGCTTTTGCCCCCTGTAGTG  47 1149 1047750 N/A N/A  4090  4109 TAGTGCCCCATCAAGAGGTA  88 1150 1047766 N/A N/A  4262  4281 TCACAAAGCCCAGCCATGAA  89 1151 1047782 N/A N/A  4319  4338 TCCAACTCCTCCTTTATATG  85 1152 1047798 N/A N/A  4357  4376 GAATCCAATCTCCCTCATGG  94 1153 1047814 N/A N/A  4396  4415 CCAGACCTGCCTGCTCTTTC  89 1154 1047830 N/A N/A  4523  4542 TCCTCTGATCCCAGGTAACC  76 1155 1047846 N/A N/A  4701  4720 TAACTCATTACTAAGGTGCC  79 1156 1047862 N/A N/A  4804  4823 GACCACCCCCACCCAGGACC 105 1157 1047878 N/A N/A  4853  4872 AGGACACTTGAATACCTGCC  56 1158 1047894 N/A N/A  5004  5023 GCCATCAATCCTTTCCTCCC 100 1159 1047910 N/A N/A  5113  5132 AGGCTCCTTCTCCCCATTCC  90 1160 1047926 N/A N/A  5379  5398 CTGCCAATCTCTGTTTCTCT  85 1161 1047942 N/A N/A  5496  5515 TTCCCCACGCCATTGTGTCC  76 1162 1047958 N/A N/A  5787  5806 TCCTTCCCCCATTCTCTTGT 121 1163 1047974 N/A N/A  5851  5870 CCATCTCACTTCTCTGGTGA  82 1164 1047990 N/A N/A  6019  6038 CGGCTCTCTCATCTGTCAAA  43 1165 1048006 N/A N/A  6115  6134 GCAGGCTCCCAAGTGAGATG  99 1166 1048022 N/A N/A  6221  6240 CGTCAATATCACACCTTCCA  96 1167 1048038 N/A N/A  6324  6343 CCTGCCTAGCCCAAATGCCC 119 1168 1048054 N/A N/A  6400  6419 GCTTTTCTGCCTCCAGGCTC  77 1169 1048070 N/A N/A  6539  6558 CCTTTCTCCCCTGCCTGCAG  89 1170 1048086 N/A N/A  6957  6976 TCCAGAACCTTCCACACTGA  82 1171 1048102 N/A N/A  7070  7089 GACTTTTCCCAACAACTGTG  84 1172 1048118 N/A N/A  7390  7409 CCCTGGCCCTTCTCCCCTGG  76 1173 1048134 N/A N/A  7842  7861 ACAAGGCTGAAAAAGACTCA  83 1174 1048150 N/A N/A  7986  8005 GAGGGTGACCCAAGTCCTTG 110 1175 1048166 N/A N/A  8221  8240 GCCAGGAAAGTCTAACTCCA  75 1176 1048182 N/A N/A  9326  9345 GTCACATTCACTAATATTTA  44 1177 1048198 N/A N/A  9525  9544 CCTCTACTAGTCAGCCTGGT  65 1178 1048214 N/A N/A  9606  9625 TCCCCTTTCCTCTTTCCTAC  75 1179 1048230 N/A N/A  9772  9791 CTCTGGGCAAGTTAATTGAC 122 1180 1048246 N/A N/A  9818  9837 ACCAGAGCTCAAGAAGTCCC  83 1181 1048262 N/A N/A 10618 10637 TGGCTTCATTTCAGCCCCTC  81 1182 1048278 N/A N/A 10710 10729 CAGAGAGAGCCTAGGCTCTT  95 1183 1048294 N/A N/A 10788 10807 TGAGCCATCCTCTCCCATGC 125 1184 1048310 N/A N/A 10940 10959 CTGGGCCCAAATCCCTCCTT  83 1185 1048326 N/A N/A 11060 11079 TGCTCTCCTCCAGAATTCCC 123 1186 1048342 N/A N/A 11223 11242 CTAACTTTAATTCTCTTTCT 114 1187 1048358 N/A N/A 11317 11336 TTCCCATTCCCCTGGTAGTT  89 1188 1048374 N/A N/A 11436 11455 GGGTGGTGAGATAACACTGG 106 1189 1048390 N/A N/A 11609 11628 TGGACTCCTTAATGACCTGC  68 1190

TABLE 17 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP RNA SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (% control) NO 1047159  126  145  3574  3593 GAGCCAGGCCCCCCACCATC 104 1191 1047175  244  263  3692  3711 CGGGTCTCCTTGAAGCCAGC 125 1192 1047191  480  499  3928  3947 CCAGATTGTCCCTCTCAACC  15* 1193 1047207  574  593 N/A N/A GCTTCCTGTCTATAGGCAGC  37* 1194 1047223  639  658  5228  5247 GGATCTCCTCCTCCAGCGAC  79 1195 1047239  811  830  5697  5716 TCTTCGGCTTCATGCATGTT  98 1196 1047255 1028 1047  7600  7619 CGCCTCCTGATAACTGGCCG 105 1197 1047271 1241 1260 10855 10874 AGACTTGGTGTCCAGGCTGG  79 1198 1047287 1289 1308 10903 10922 CTCCACGGTCTTCACCACGA 138 1199 1047303 1349 1368 11638 11657 GCCTCACATCACATCCTTGT 113 1200 1047319 1414 1433 11703 11722 GAGCAACTATCCTGCTTCTG  68 1201 1047335 1615 1634 11904 11923 GCTGCTGGCCATGCCCCTCC 123 1202 1047351 1663 1682 11952 11971 GCCCCCCGCCCTCCTCCCCT  73 1203 1047367 1734 1753 12023 12042 GAGAACCTCCATCTCTGGCA  67 1204 1047383 1790 1809 12079 12098 CAGTTTTCCTCCAGCAGCCT 109   11 1047399 1853 1872 12142 12161 ACAAAACAAGCCTCTGGCCA  90 1206 1047415 2035 2054 12324 12343 GTCCCCACCCATCTTAGACT  76 1207 1047431 2179 2198 12468 12487 TGCTGAGAATCAAGCTCCCA  91 1208 1047447 2236 2255 12525 12544 CCCCTCTATCCCTCCCAGCA  83 1209 1047463 2279 2298 12568 12587 TGGGCTTGACCTCTCTGTAC 103 1210 1047479 2377 2396 12666 12685 GTCACCCACAACCCCTACTT  88 1211 1047495 2456 2475 12745 12764 CCTTTCTCTCCTGTTTCAGC  54 1212 1047511 2482 2501 12771 12790 CATGCCCTGCCCCCATGGAT  83 1213 1047527 2779 2798 13068 13087 CACCCGGCCTCCAGGCTGCA 106 1214 1047543 2860 2879 13149 13168 GCACAGATCCCACCAGTCTG 108 1215 1047559 2902 2921 13191 13210 AGAGGAGAACCCTGAAGTGG 120 1216 1047575 3034 3053 13323 13342 CTCAGCGACTAAAGGCAGCA  59 1217 1047591 3056 3075 13345 13364 GCAGCATTTGTCTTTATTTT  22 1218 1047607 N/A N/A  8760  8779 TGACCTTGTGATTTTCCCCG  59 1219 1047623 N/A N/A  9025  9044 TGTACAGTTACTCTGTACCA 103 1220 1047639 N/A N/A  9067  9086 AGGACTCACCACCTTTACCA  77 1221 1047655 N/A N/A  9201  9220 GGGATGAAAGAATAAAGCAG  67 1222 1047671 N/A N/A  8378  8397 GCTGTACTGACCTCGAATCT  94 1223 1047687 N/A N/A  8453  8472 CTCAGTCCCAGTCTGGAGCA 123 1224 1047703 N/A N/A  8502  8521 GCAGTGTCACGAAGGCCCCC  89 1225 1047719 N/A N/A  8635  8654 TCAAGCTCTCACCCAGTTCT  96 1226 1047735 N/A N/A  8735  8754 GGTGCTTTTGCCCCCTGTAG  55 1227 1047751 N/A N/A  4091  4110 ATAGTGCCCCATCAAGAGGT 135 1228 1047767 N/A N/A  4263  4282 GTCACAAAGCCCAGCCATGA 122 1229 1047783 N/A N/A  4321  4340 CTTCCAACTCCTCCTTTATA  95 1230 1047799 N/A N/A  4358  4377 AGAATCCAATCTCCCTCATG  67 1231 1047815 N/A N/A  4399  4418 CGCCCAGACCTGCCTGCTCT  94 1232 1047831 N/A N/A  4524  4543 TTCCTCTGATCCCAGGTAAC  86 1233 1047847 N/A N/A  4702  4721 TTAACTCATTACTAAGGTGC  87 1234 1047863 N/A N/A  4805  4824 AGACCACCCCCACCCAGGAC  96 1235 1047879 N/A N/A  4866  4885 CCAGGCTCTTCTGAGGACAC 119 1236 1047895 N/A N/A  5005  5024 GGCCATCAATCCTTTCCTCC  81 1237 1047911 N/A N/A  5115  5134 CCAGGCTCCTTCTCCCCATT  92 1238 1047927 N/A N/A  5385  5404 CTCTACCTGCCAATCTCTGT  86 1239 1047943 N/A N/A  5497  5516 GTTCCCCACGCCATTGTGTC  78 1240 1047959 N/A N/A  5788  5807 CTCCTTCCCCCATTCTCTTG 104 1241 1047975 N/A N/A  5933  5952 GCTACTACTAATAATAGCAA  99 1242 1047991 N/A N/A  6021  6040 TTCGGCTCTCTCATCTGTCA  80 1243 1048007 N/A N/A  6117  6136 ATGCAGGCTCCCAAGTGAGA  88 1244 1048023 N/A N/A  6280  6299 CCACACTACATATAAGCTCT 163 1245 1048039 N/A N/A  6325  6344 TCCTGCCTAGCCCAAATGCC 100 1246 1048055 N/A N/A  6403  6422 TGTGCTTTTCTGCCTCCAGG  52 1247 1048071 N/A N/A  6543  6562 TAGCCCTTTCTCCCCTGCCT  77 1248 1048087 N/A N/A  6958  6977 ATCCAGAACCTTCCACACTG  88 1249 1048103 N/A N/A  7072  7091 GGGACTTTTCCCAACAACTG  68 1250 1048119 N/A N/A  7393  7412 CGTCCCTGGCCCTTCTCCCC  77 1251 1048135 N/A N/A  7843  7862 CACAAGGCTGAAAAAGACTC  94 1252 1048151 N/A N/A  7987  8006 GGAGGGTGACCCAAGTCCTT  35 1253 1048167 N/A N/A  8222  8241 TGCCAGGAAAGTCTAACTCC  77 1254 1048183 N/A N/A  9362  9381 TCCCCCCGCCCCGCCCGAGA  88 1255 1048199 N/A N/A  9533  9552 CAGTATTACCTCTACTAGTC  64   20 1048215 N/A N/A  9609  9628 CTGTCCCCTTTCCTCTTTCC  98 1256 1048231 N/A N/A  9788  9807 CCAACCAGCCACATGACTCT  91 1257 1048247 N/A N/A  9825  9844 TCAGGAGACCAGAGCTCAAG  93 1258 1048263 N/A N/A 10620 10639 CCTGGCTTCATTTCAGCCCC  87 1259 1048279 N/A N/A 10711 10730 GCAGAGAGAGCCTAGGCTCT 143 1260 1048295 N/A N/A 10792 10811 GGCATGAGCCATCCTCTCCC 123 1261 1048311 N/A N/A 10941 10960 ACTGGGCCCAAATCCCTCCT 104 1262 1048327 N/A N/A 11061 11080 TTGCTCTCCTCCAGAATTCC  97 1263 1048343 N/A N/A 11226 11245 CTACTAACTTTAATTCTCTT  97 1264 1048359 N/A N/A 11318 11337 CTTCCCATTCCCCTGGTAGT  86 1265 1048375 N/A N/A 11487 11506 GTCTTACTTTTCTTGATAGT  94 1266 1048391 N/A N/A 11610 11629 TTGGACTCCTTAATGACCTG  86 1267

Example 2: Effect of 5-10-5 MOE Gapmer Modified Oligonucleotides on Human GFAP RNA In Vitro, Single Dose

Modified oligonucleotides complementary to human GFAP nucleic acid were designed and tested for their single dose effects on GFAP RNA in vitro. The modified oligonucleotides were tested in a series of experiments that had similar culture conditions.

The modified oligonucleotides in the tables below are 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages. The gapmers are 20 nucleosides in length, wherein the central gap segment consists of ten 2′-β-D-deoxynucleosides and the 5′ and 3′ wing segments each consists of five 2′-β-D-MOE modified nucleosides. The sugar motif for the gapmers is (from 5′ to 3′): eeeeeddddddddddeeeee; wherein ‘d’ represents a 2′-β-D-deoxyribosyl sugar moiety, and ‘e’ represents a 2′-β-D-MOE sugar moiety. The internucleoside linkage motif for the gapmers is (from 5′ to 3′): sooosssssssssssooss; wherein each ‘o’ represents a phosphodiester internucleoside linkage and each ‘s’ represents a phosphorothioate internucleoside linkage. Each cytosine residue is a 5-methyl cytosine.

“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is complementary in the target nucleic acid sequence. Each modified oligonucleotide listed in the Tables below is 100% complementary to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 (GENBANK Accession No. NM_001131019.2). ‘N/A’ indicates that the modified oligonucleotide is not 100% complementary to that particular gene sequence.

Cultured U251 cells were treated with modified oligonucleotide at a concentration of 4,000 nM using free uptake at a density of 10,000 cells per well. After a treatment period of approximately 48 hours, total RNA was isolated from the cells and GFAP RNA levels were measured by quantitative real-time RTPCR. Human GFAP primer probe set RTS37485, described in Example 1 above, was used to measure RNA levels. GFAP RNA levels were normalized to total RNA content, as measured by RIBOGREEN®. Results are presented in the tables below as percent GFAP RNA levels relative to untreated control cells. The values marked with an asterisk (*) indicate that the modified oligonucleotide is complementary to the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of the modified oligonucleotides complementary to the amplicon region.

TABLE 18 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 Compound Start Stop Start Stop GFAP SEQ ID No. Site Site Site Site Sequence (5′ to 3′) (%UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG   8  517 1072810 1708 1727 11997 12016 TCCATAACAACAGGAATCAG  65 1268 1072814 1719 1738 12008 12027 TGGCAACAGTTTCCATAACA  31 1269 1072818 1728 1747 12017 12036 CTCCATCTCTGGCAACAGTT  42 1270 1072822 1752 1771 12041 12060 AGTTCCCAGATACTCCGAGA  56 1271 1072826 1763 1782 12052 12071 CTCAAAGGCACAGTTCCCAG  56 1272 1072830 1786 1805 12075 12094 TTTCCTCCAGCAGCCTGAGG  79 1273 1072834 1796 1815 12085 12104 GAGTCTCAGTTTTCCTCCAG  18 1274 1072838 2173 2192 12462 12481 GAATCAAGCTCCCACCTGCC  87 1275 1072842 2177 2196 12466 12485 CTGAGAATCAAGCTCCCACC  94 1276 1072846 3039 3058 13328 13347 TTTTCCTCAGCGACTAAAGG  48 1277 1072850 3059 3078 13348 13367 GGCGCAGCATTTGTCTTTAT  45 1278 1072854 N/A N/A  8768  8787 ATATCTTGTGACCTTGTGAT  53 1279 1072858 N/A N/A  8774  8793 TTTGAGATATCTTGTGACCT  66 1280 1072862 N/A N/A  8780  8799 GAGGCTTTTGAGATATCTTG  28 1281 1072866 N/A N/A  8785  8804 ATTGTGAGGCTTTTGAGATA  66 1282 1072870 N/A N/A  7980  7999 GACCCAAGTCCTTGGCCTTG  81 1283 1072874 N/A N/A  7990  8009 TTTGGAGGGTGACCCAAGTC  78 1284 1072878 N/A N/A  7997  8016 CTCTTAGTTTGGAGGGTGAC  61 1285 1072882 N/A N/A 11296 11315 CCTTTTACCAAGCTGGAAAT  73 1286 1072886 N/A N/A 11303 11322 GTAGTTTCCTTTTACCAAGC  34 1287 1072890 N/A N/A  4033  4052 GAGACTTCTCGGGCACTCCT  75 1288 1072894 N/A N/A  4133  4152 TGGGCCTGTTTCTGGTCCCT  63 1289 1072898 N/A N/A  4208  4227 TTCCCCAGTAGGGAGGTGCT 100 1290 1072902 N/A N/A  4285  4304 ATAGGTGAGCTCGCTGCCCA  82 1291 1072906 N/A N/A  4297  4316 CACAGGCTCAGAATAGGTGA  68 1292 1072910 N/A N/A  4458  4477 CAAGTCAAAGTAACTTGATG  80 1293 1072914 N/A N/A  4492  4511 TGAATTTTATTATGACCACC  64 1294 1072918 N/A N/A  4564  4583 CATGTCCTGTCAGCTCAGTG  61 1295 1072922 N/A N/A  4638  4657 CACAAGCATACACTCACTGT  84 1296 1072926 N/A N/A  4677  4696 CAGGGTTGGTGCACCTGCTT  74 1297 1072930 N/A N/A  4748  4767 TAGACAGAGGACTTGTCTGG  96 1298 1072934 N/A N/A  4826  4845 CCCTTGAGGCAGCTGTCACA  87 1299 1072938 N/A N/A  5049  5068 CATTGCTCTGGCGGGCTGAG  89 1300 1072942 N/A N/A  5296  5315 CAATCTCTGTGTTGAGCTTT  70 1301 1072946 N/A N/A  5396  5415 TCATTTCCTGTCTCTACCTG  85 1302 1072950 N/A N/A  5549  5568 GACCAGGGTGAGAGAAGCGG  78 1303 1072954 N/A N/A  5745  5764 GAGGAGGCAGGCTGGCCCAC  84 1304 1072958 N/A N/A  5900  5919 AATAATGGGTACTTTTGAAA  88 1305 1072962 N/A N/A  5986  6005 TTCATAGTAAGGTAATCCAT  75 1306 1072966 N/A N/A  6032  6051 CCTCTCTGGACTTCGGCTCT  81 1307 1072970 N/A N/A  6240  6259 GGCACTATGTTTGGGTGCAC  85 1308 1072974 N/A N/A  6302  6321 TCTACAGTGTCTTTCCTGGC  67 1309 1072978 N/A N/A  6446  6465 CTAGGTGCCCTGGCTAGGCT  72 1310 1072982 N/A N/A  6524  6543 TGCAGGGAGGTCCTCCCAGC  96 1311 1072986 N/A N/A  6901  6920 GCGAGCGGAGGCCTGGGTGT  26 1312 1072990 N/A N/A  6942  6961 ACTGACAGCTGCATCTGCGG  79 1313 1072994 N/A N/A  6985  7004 AAGCGAATGAATGAACAGTG  69 1314 1072998 N/A N/A  7079  7098 CCTGGCTGGGACTTTTCCCA  84 1315 1073002 N/A N/A  7119  7138 GGGAGGTGAGCAGCACCCCA  84 1316 1073006 N/A N/A  7358  7377 TGGCCGTCCCTGCTCCGCCC  96 1317 1073010 N/A N/A  7510  7529 GGCCGGTCCCGCGGAGCCCC  86 1318 1073014 N/A N/A  7521  7540 GGGATGGAGCCGGCCGGTCC  77 1319 1073018 N/A N/A  7785  7804 AGCAGGGAGACTTCCCCAGG  85 1320 1073022 N/A N/A  7827  7846 ACTCAGTCCCTGAAGGGAGC  90 1321 1073026 N/A N/A  7898  7917 CTGCTATGTGTGAGGCAGGC  83 1322 1073030 N/A N/A  8027  8046 CAATCTTGGCTGGGAAGATG  90 1323 1073034 N/A N/A  8048  8067 AGATGGGTGAGGTGAGGAGT  33 1324 1073038 N/A N/A  8231  8250 CCTTTTCCTTGCCAGGAAAG  76 1325 1073042 N/A N/A  9380  9399 AGTAATTTAGCTCCCCCCTC  76 1326 1073046 N/A N/A  9410  9429 AGAATCATTTCAGGGCCAAT  67 1327 1073050 N/A N/A  9438  9457 GAAGAAGAGGAATTTTGTTC  82 1328 1073054 N/A N/A  9486  9505 TTAAGTCCTGAGACATGCAT  55 1329 1073058 N/A N/A  9500  9519 TTTCTGATGCTGAATTAAGT  88 1330 1073062 N/A N/A  9543  9562 TAGGATTTGGCAGTATTACC  62 1331 1073066 N/A N/A  9649  9668 TGTGGCACATATTAGTGCTC  76 1332 1073070 N/A N/A 10001 10020 AATCCCCTTACTCGGGAGTC  76 1333 1073074 N/A N/A 10551 10570 TTGAAATCAGGAGACCAGGA  73 1334 1073078 N/A N/A 10567 10586 AAAACCCAGCACGGTATTGA  73 1335 1073082 N/A N/A 10715 10734 CCGAGCAGAGAGAGCCTAGG  88 1336 1073086 N/A N/A 10805 10824 CATGGACTTTCAGGGCATGA  90 1337 1073090 N/A N/A 11105 11124 CAGCCTATGGAGGGACTGAG  88 1338 1073094 N/A N/A 11165 11184 AAGAGAGAGTGTGTATTAGG  63 1339 1073098 N/A N/A 11208 11227 TTTCTCTCCCTGGCAAGCAA  65 1340 1073102 N/A N/A 11256 11275 AACTGTGTCTGCTAGAGTTG  66 1341 1073106 N/A N/A 11386 11405 GTAAGCTGCTGGAGTAAGAT  45 1342 1073110 N/A N/A 11472 11491 ATAGTAACCACAGCTGCCTT  81 1343 1073114 N/A N/A 11516 11535 GTAACCTTGGGAAGTCCCCG  75 1344

TABLE 19 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG 7 517 1072811 1711 1730 12000 12019 GTTTCCATAACAACAGGAAT 82 1345 1072815 1720 1739 12009 12028 CTGGCAACAGTTTCCATAAC 40 1346 1072819 1746 1765 12035 12054 CAGATACTCCGAGAGAACCT 75 1347 1072823 1753 1772 12042 12061 CAGTTCCCAGATACTCCGAG 65 1348 1072827 1766 1785 12055 12074 AAACTCAAAGGCACAGTTCC 99 1349 1072831 1787 1806 12076 12095 TTTTCCTCCAGCAGCCTGAG 85 1350 1072835 1797 1816 12086 12105 TGAGTCTCAGTTTTCCTCCA 18 1351 1072839 2174 2193 12463 12482 AGAATCAAGCTCCCACCTGC 82 1352 1072843 3036 3055 13325 13344 TCCTCAGCGACTAAAGGCAG 54 1353 1072847 3041 3060 13330 13349 ATTTTTCCTCAGCGACTAAA 55 1354 1072851 3061 3080 13350 13369 AGGGCGCAGCATTTGTCTTT 55 1355 1072855 N/A N/A 8771 8790 GAGATATCTTGTGACCTTGT 37 1356 1072859 N/A N/A 8775 8794 TTTTGAGATATCTTGTGACC 60 1357 1072863 N/A N/A 8781 8800 TGAGGCTTTTGAGATATCTT 34 1358 1072867 N/A N/A 8786 8805 TATTGTGAGGCTTTTGAGAT 60 1359 1072871 N/A N/A 7983 8002 GGTGACCCAAGTCCTTGGCC 76 1360 1072875 N/A N/A 7991 8010 GTTTGGAGGGTGACCCAAGT 70 1361 1072879 N/A N/A 11290 11309 ACCAAGCTGGAAATGGAAAG 70 1362 1072883 N/A N/A 11297 11316 TCCTTTTACCAAGCTGGAAA 83 1363 1072887 N/A N/A 11304 11323 GGTAGTTTCCTTTTACCAAG 49 1364 1072891 N/A N/A 4035 4054 CTGAGACTTCTCGGGCACTC 87 1365 1072895 N/A N/A 4162 4181 GGCATGCGGGCATCAGATCC 87 1366 1072899 N/A N/A 4221 4240 CTCCTGCACTGCTTTCCCCA 64 1367 1072903 N/A N/A 4287 4306 GAATAGGTGAGCTCGCTGCC 89 1368 1072907 N/A N/A 4304 4323 ATATGGACACAGGCTCAGAA 79 1369 1072911 N/A N/A 4463 4482 CTGTGCAAGTCAAAGTAACT 76 1370 1072915 N/A N/A 4493 4512 ATGAATTTTATTATGACCAC 67 1371 1072919 N/A N/A 4581 4600 ACTTGAAGGCACACATGCAT 70 1372 1072923 N/A N/A 4648 4667 CAGGCGCATCCACAAGCATA 88 1373 1072927 N/A N/A 4732 4751 CTGGAGGATGAGCAGATGTG 57 1374 1072931 N/A N/A 4779 4798 AGCAGCAGGAGGATTAAGGG 68 1375 1072935 N/A N/A 4827 4846 TCCCTTGAGGCAGCTGTCAC 96 1376 1072939 N/A N/A 5058 5077 GGAGCAGCACATTGCTCTGG 80 1377 1072943 N/A N/A 5299 5318 TTGCAATCTCTGTGTTGAGC 55 1378 1072947 N/A N/A 5452 5471 AGTTCGAATGCTCTCTTGTC 78 1379 1072951 N/A N/A 5715 5734 ACCTTGGAGCGGTACCACTC 111 1380 1072955 N/A N/A 5828 5847 TCAGTCACCTGGAGAGGATA 58 1381 1072959 N/A N/A 5918 5937 AGCAATAGTAGCAGTAATAA 82 1382 1072963 N/A N/A 5987 6006 CTTCATAGTAAGGTAATCCA 84 1383 1072967 N/A N/A 6122 6141 ATGGAATGCAGGCTCCCAAG 71 1384 1072971 N/A N/A 6251 6270 TGTTCTCTACGGGCACTATG 58 1385 1072975 N/A N/A 6374 6393 CCACTAGGAATGGCCCTCCC 61 1386 1072979 N/A N/A 6451 6470 CTCAGCTAGGTGCCCTGGCT 80 1387 1072983 N/A N/A 6561 6580 CCTCACCCTGGGTTCTAATA 83 1388 1072987 N/A N/A 6903 6922 AGGCGAGCGGAGGCCTGGGT 97 1389 1072991 N/A N/A 6944 6963 ACACTGACAGCTGCATCTGC 79 1390 1072995 N/A N/A 6997 7016 CACCTGGTCAGCAAGCGAAT 78 1391 1072999 N/A N/A 7082 7101 GGCCCTGGCTGGGACTTTTC 79 1392 1073003 N/A N/A 7125 7144 AAATCAGGGAGGTGAGCAGC 46 1393 1073007 N/A N/A 7382 7401 CTTCTCCCCTGGCATCTCCT 77 1394 1073011 N/A N/A 7517 7536 TGGAGCCGGCCGGTCCCGCG 87 1395 1073015 N/A N/A 7757 7776 TGAGGGCTCACCGGTTCTCC 81 1396 1073019 N/A N/A 7789 7808 AGGCAGCAGGGAGACTTCCC 88 1397 1073023 N/A N/A 7855 7874 AAGGGATCTGCACACAAGGC 79 1398 1073027 N/A N/A 7924 7943 TCAGTCATCAAACATCTAGT 78 1399 1073031 N/A N/A 8028 8047 CCAATCTTGGCTGGGAAGAT 66 1400 1073035 N/A N/A 8051 8070 AAGAGATGGGTGAGGTGAGG 36 1401 1073039 N/A N/A 8274 8293 TAGGCTGGGTCTTGGTGCGG 76 1402 1073043 N/A N/A 9381 9400 AAGTAATTTAGCTCCCCCCT 76 1403 1073047 N/A N/A 9411 9430 AAGAATCATTTCAGGGCCAA 54 1404 1073051 N/A N/A 9440 9459 CAGAAGAAGAGGAATTTTGT 70 1405 1073055 N/A N/A 9487 9506 ATTAAGTCCTGAGACATGCA 64 1406 1073059 N/A N/A 9502 9521 CCTTTCTGATGCTGAATTAA 72 1407 1073063 N/A N/A 9552 9571 GTGACTATCTAGGATTTGGC 23 1408 1073067 N/A N/A 9685 9704 GAGGAGACAATTAACTAAAA 64 1409 1073071 N/A N/A 10125 10144 TCGAAAGCAGGCAAGCAAGC 99 1410 1073075 N/A N/A 10552 10571 ATTGAAATCAGGAGACCAGG 66 1411 1073079 N/A N/A 10612 10631 CATTTCAGCCCCTCTGCAAG 77 1412 1073083 N/A N/A 10724 10743 CCTATGCAACCGAGCAGAGA 95 1413 1073087 N/A N/A 10848 10867 GTGTCCAGGCTGGTTTCTGC 82 1414 1073091 N/A N/A 11124 11143 ACCAAGGCTCCCCTTAGAAC 59 1415 1073095 N/A N/A 11166 11185 AAAGAGAGAGTGTGTATTAG 78 1416 1073099 N/A N/A 11210 11229 TCTTTCTCTCCCTGGCAAGC 73 1417 1073103 N/A N/A 11283 11302 TGGAAATGGAAAGCCCTCCC 78 1418 1073107 N/A N/A 11391 11410 GAGTGGTAAGCTGCTGGAGT 42 1419 1073111 N/A N/A 11474 11493 TGATAGTAACCACAGCTGCC 92 1420 1073115 N/A N/A 11518 11537 GTGTAACCTTGGGAAGTCCC 77 1421

TABLE 20 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG 7 517 1072812 1713 1732 12002 12021 CAGTTTCCATAACAACAGGA 58 1422 1072816 1722 1741 12011 12030 CTCTGGCAACAGTTTCCATA 69 1423 1072820 1749 1768 12038 12057 TCCCAGATACTCCGAGAGAA 73 1424 1072824 1758 1777 12047 12066 AGGCACAGTTCCCAGATACT 36 1425 1072828 1784 1803 12073 12092 TCCTCCAGCAGCCTGAGGAA 71 1426 1072832 1788 1807 12077 12096 GTTTTCCTCCAGCAGCCTGA 64 1427 1072836 2168 2187 12457 12476 AAGCTCCCACCTGCCCACAG 73 1428 1072840 2175 2194 12464 12483 GAGAATCAAGCTCCCACCTG 74 1429 1072844 3037 3056 13326 13345 TTCCTCAGCGACTAAAGGCA 61 1430 1072848 3042 3061 13331 13350 TATTTTTCCTCAGCGACTAA 69 1431 1072852 3063 3082 13352 13371 GAAGGGCGCAGCATTTGTCT 57 1432 1072856 N/A N/A 8772 8791 TGAGATATCTTGTGACCTTG 43 1433 1072860 N/A N/A 8776 8795 CTTTTGAGATATCTTGTGAC 62 1434 1072864 N/A N/A 8783 8802 TGTGAGGCTTTTGAGATATC 43 1435 1072868 N/A N/A 8788 8807 CGTATTGTGAGGCTTTTGAG 20 1436 1072872 N/A N/A 7984 8003 GGGTGACCCAAGTCCTTGGC 14 1437 1072876 N/A N/A 7992 8011 AGTTTGGAGGGTGACCCAAG 61 1438 1072880 N/A N/A 11293 11312 TTTACCAAGCTGGAAATGGA 83 1439 1072884 N/A N/A 11298 11317 TTCCTTTTACCAAGCTGGAA 77 1440 1072888 N/A N/A 11310 11329 TCCCCTGGTAGTTTCCTTTT 85 1441 1072892 N/A N/A 4055 4074 CAGGGAGGTTCGGCCCCTCC 83 1442 1072896 N/A N/A 4174 4193 CTCCTGGCAGAAGGCATGCG 75 1443 1072900 N/A N/A 4231 4250 GGCCCCGCTGCTCCTGCACT 105 1444 1072904 N/A N/A 4289 4308 CAGAATAGGTGAGCTCGCTG 83 1445 1072908 N/A N/A 4307 4326 TTTATATGGACACAGGCTCA 83 1446 1072912 N/A N/A 4482 4501 TATGACCACCGCTTCACAGC 95 1447 1072916 N/A N/A 4558 4577 CTGTCAGCTCAGTGAAGCGC 86 1448 1072920 N/A N/A 4628 4647 CACTCACTGTTGCACACACA 89 1449 1072924 N/A N/A 4651 4670 ACACAGGCGCATCCACAAGC 98 1450 1072928 N/A N/A 4739 4758 GACTTGTCTGGAGGATGAGC 73 1451 1072932 N/A N/A 4785 4804 CAGTAGAGCAGCAGGAGGAT 82 1452 1072936 N/A N/A 4882 4901 GGACACATTCCTGGGTCCAG 100 1453 1072940 N/A N/A 5142 5161 GGTGAGGAGTAGAGGGCCAC 88 1454 1072944 N/A N/A 5307 5326 TCTCTCAGTTGCAATCTCTG 84 1455 1072948 N/A N/A 5453 5472 GAGTTCGAATGCTCTCTTGT 75 1456 1072952 N/A N/A 5730 5749 CCCACAGGCAGGGCTACCTT 89 1457 1072956 N/A N/A 5891 5910 TACTTTTGAAAGCAATAGTG 77 1458 1072960 N/A N/A 5953 5972 CTTAGAACAGAACAGTATCA 101 1459 1072964 N/A N/A 6028 6047 TCTGGACTTCGGCTCTCTCA 55 1460 1072968 N/A N/A 6140 6159 AAACAGACTGGCAGAGGCAT 59 1461 1072972 N/A N/A 6260 6279 GAGCTGTGGTGTTCTCTACG 58 1462 1072976 N/A N/A 6380 6399 TGTCCTCCACTAGGAATGGC 76 1463 1072980 N/A N/A 6459 6478 TCACACTCCTCAGCTAGGTG 32 1464 1072984 N/A N/A 6563 6582 GGCCTCACCCTGGGTTCTAA 76 1465 1072988 N/A N/A 6905 6924 TTAGGCGAGCGGAGGCCTGG 95 1466 1072992 N/A N/A 6945 6964 CACACTGACAGCTGCATCTG 90 1467 1072996 N/A N/A 7003 7022 GCACAACACCTGGTCAGCAA 73 1468 1073000 N/A N/A 7085 7104 GTTGGCCCTGGCTGGGACTT 63 1469 1073004 N/A N/A 7126 7145 GAAATCAGGGAGGTGAGCAG 63 1470 1073008 N/A N/A 7475 7494 GGTTTCGAGGCCCGGCCCCC 73 1471 1073012 N/A N/A 7518 7537 ATGGAGCCGGCCGGTCCCGC 92 1472 1073016 N/A N/A 7762 7781 TGTGATGAGGGCTCACCGGT 65 1473 1073020 N/A N/A 7797 7816 CTACCGTGAGGCAGCAGGGA 86 1474 1073024 N/A N/A 7876 7895 TGTGCTGGGCATTGAGGTGG 64 1475 1073028 N/A N/A 7967 7986 GGCCTTGAGGCCTAATCAAT 78 1476 1073032 N/A N/A 8045 8064 TGGGTGAGGTGAGGAGTCCA 73 1477 1073036 N/A N/A 8057 8076 AGGCAGAAGAGATGGGTGAG 67 1478 1073040 N/A N/A 9355 9374 GCCCCGCCCGAGAGAGAAAA 92 1479 1073044 N/A N/A 9407 9426 ATCATTTCAGGGCCAATGCA 66 1480 1073048 N/A N/A 9423 9442 TGTTCCTTAGCTAAGAATCA 58 1481 1073052 N/A N/A 9442 9461 TCCAGAAGAAGAGGAATTTT 94 1482 1073056 N/A N/A 9488 9507 AATTAAGTCCTGAGACATGC 51 1483 1073060 N/A N/A 9517 9536 AGTCAGCCTGGTTAGCCTTT 36 1484 1073064 N/A N/A 9560 9579 AGTGACCTGTGACTATCTAG 35 1485 1073068 N/A N/A 9889 9908 TCTGCCGAAGGAAGGAAGGA 77 1486 1073072 N/A N/A 10129 10148 TCCGTCGAAAGCAGGCAAGC 83 1487 1073076 N/A N/A 10555 10574 GGTATTGAAATCAGGAGACC 59 1488 1073080 N/A N/A 10662 10681 CAATAGTGCTGCTGCCAGAG 54 1489 1073084 N/A N/A 10727 10746 GAACCTATGCAACCGAGCAG 56 1490 1073088 N/A N/A 11008 11027 TATGTGCCAGCCCCAGGCTT 89 1491 1073092 N/A N/A 11128 11147 TGGTACCAAGGCTCCCCTTA 74 1492 1073096 N/A N/A 11187 11206 GCCCCCGAGTTTGAGGGTGA 90 1493 1073100 N/A N/A 11240 11259 GTTGGAAGTGAAAGCTACTA 58 1494 1073104 N/A N/A 11365 11384 AGCTCCCACTGTGGTTGGAG 87 1495 1073108 N/A N/A 11399 11418 TGCCTGGCGAGTGGTAAGCT 91 1496 1073112 N/A N/A 11476 11495 CTTGATAGTAACCACAGCTG 63 1497 1073116 N/A N/A 11561 11580 AGTTAGGAGTTCACACAGAC 72 1498

TABLE 21 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG 8 517 1072813 1717 1736 12006 12025 GCAACAGTTTCCATAACAAC 32 1499 1072817 1725 1744 12014 12033 CATCTCTGGCAACAGTTTCC 62 1500 1072821 1751 1770 12040 12059 GTTCCCAGATACTCCGAGAG 53 1501 1072825 1759 1778 12048 12067 AAGGCACAGTTCCCAGATAC 51 1502 1072829 1785 1804 12074 12093 TTCCTCCAGCAGCCTGAGGA 89 1503 1072833 1789 1808 12078 12097 AGTTTTCCTCCAGCAGCCTG 46 1504 1072837 2171 2190 12460 12479 ATCAAGCTCCCACCTGCCCA 64 1505 1072841 2176 2195 12465 12484 TGAGAATCAAGCTCCCACCT 90 1506 1072845 3038 3057 13327 13346 TTTCCTCAGCGACTAAAGGC 52 1507 1072849 3057 3076 13346 13365 CGCAGCATTTGTCTTTATTT 26 1508 1072853 3064 3083 13353 13372 GGAAGGGCGCAGCATTTGTC 43 1509 1072857 N/A N/A 8773 8792 TTGAGATATCTTGTGACCTT 32 1510 1072861 N/A N/A 8779 8798 AGGCTTTTGAGATATCTTGT 35 1511 1072865 N/A N/A 8784 8803 TTGTGAGGCTTTTGAGATAT 47 1512 1072869 N/A N/A 7977 7996 CCAAGTCCTTGGCCTTGAGG 93 1513 1072873 N/A N/A 7988 8007 TGGAGGGTGACCCAAGTCCT 107 1514 1072877 N/A N/A 7994 8013 TTAGTTTGGAGGGTGACCCA 68 1515 1072881 N/A N/A 11295 11314 CTTTTACCAAGCTGGAAATG 103 1516 1072885 N/A N/A 11299 11318 TTTCCTTTTACCAAGCTGGA 80 1517 1072889 N/A N/A 4027 4046 TCTCGGGCACTCCTTCTTGG 96 1518 1072893 N/A N/A 4084 4103 CCCATCAAGAGGTAGGGAGG 66 1519 1072897 N/A N/A 4183 4202 GACCCTGGACTCCTGGCAGA 83 1520 1072901 N/A N/A 4253 4272 CCAGCCATGAATGAAACACA 82 1521 1072905 N/A N/A 4293 4312 GGCTCAGAATAGGTGAGCTC 67 1522 1072909 N/A N/A 4435 4454 GTCACAAGCTGGTGGCAGGC 69 1523 1072913 N/A N/A 4487 4506 TTTATTATGACCACCGCTTC 92 1524 1072917 N/A N/A 4563 4582 ATGTCCTGTCAGCTCAGTGA 50 1525 1072921 N/A N/A 4632 4651 CATACACTCACTGTTGCACA 83 1526 1072925 N/A N/A 4669 4688 GTGCACCTGCTTCTGCTCAC 111 1527 1072929 N/A N/A 4741 4760 AGGACTTGTCTGGAGGATGA 47 1528 1072933 N/A N/A 4822 4841 TGAGGCAGCTGTCACAGAGA 102 1529 1072937 N/A N/A 4904 4923 CATCCTGGAGCCTGGAGTGG 87 1530 1072941 N/A N/A 5295 5314 AATCTCTGTGTTGAGCTTTC 56 1531 1072945 N/A N/A 5308 5327 CTCTCTCAGTTGCAATCTCT 75 1532 1072949 N/A N/A 5460 5479 CGGCCAGGAGTTCGAATGCT 81 1533 1072953 N/A N/A 5733 5752 TGGCCCACAGGCAGGGCTAC 83 1534 1072957 N/A N/A 5895 5914 TGGGTACTTTTGAAAGCAAT 75 1535 1072961 N/A N/A 5965 5984 AAAAGCACAGGGCTTAGAAC 76 1536 1072965 N/A N/A 6031 6050 CTCTCTGGACTTCGGCTCTC 88 1537 1072969 N/A N/A 6167 6186 GGCATATGGTAGAGGCTCAG 56 1538 1072973 N/A N/A 6270 6289 TATAAGCTCTGAGCTGTGGT 37 1539 1072977 N/A N/A 6443 6462 GGTGCCCTGGCTAGGCTAGC 77 1540 1072981 N/A N/A 6460 6479 CTCACACTCCTCAGCTAGGT 77 1541 1072985 N/A N/A 6897 6916 GCGGAGGCCTGGGTGTTTTG 75 1542 1072989 N/A N/A 6908 6927 GTCTTAGGCGAGCGGAGGCC 90 1543 1072993 N/A N/A 6972 6991 AACAGTGCCACAGAATCCAG 91 1544 1072997 N/A N/A 7051 7070 GACCCATGGATGCGGGCAGG 68 1545 1073001 N/A N/A 7086 7105 CGTTGGCCCTGGCTGGGACT 71 1546 1073005 N/A N/A 7201 7220 TCTGTCAGGTCTGCAAACTA 83 1547 1073009 N/A N/A 7479 7498 GGGAGGTTTCGAGGCCCGGC 41 1548 1073013 N/A N/A 7520 7539 GGATGGAGCCGGCCGGTCCC 81 1549 1073017 N/A N/A 7764 7783 GCTGTGATGAGGGCTCACCG 68 1550 1073021 N/A N/A 7818 7837 CTGAAGGGAGCAAGATGAGC 82 1551 1073025 N/A N/A 7878 7897 ACTGTGCTGGGCATTGAGGT 77 1552 1073029 N/A N/A 8005 8024 GAGTATGCCTCTTAGTTTGG 63 1553 1073033 N/A N/A 8046 8065 ATGGGTGAGGTGAGGAGTCC 36 1554 1073037 N/A N/A 8078 8097 GTGGTGAAGAAAGTTCCAAG 88 1555 1073041 N/A N/A 9356 9375 CGCCCCGCCCGAGAGAGAAA 64 1556 1073045 N/A N/A 9409 9428 GAATCATTTCAGGGCCAATG 27 1557 1073049 N/A N/A 9425 9444 TTTGTTCCTTAGCTAAGAAT 62 1558 1073053 N/A N/A 9444 9463 AGTCCAGAAGAAGAGGAATT 68 1559 1073057 N/A N/A 9497 9516 CTGATGCTGAATTAAGTCCT 50 1560 1073061 N/A N/A 9519 9538 CTAGTCAGCCTGGTTAGCCT 57 1561 1073065 N/A N/A 9577 9596 CCATTTATCTGTGCTTTAGT 39 1562 1073069 N/A N/A 9892 9911 CGCTCTGCCGAAGGAAGGAA 61 1563 1073073 N/A N/A 10548 10567 AAATCAGGAGACCAGGAGGG 59 1564 1073077 N/A N/A 10561 10580 CAGCACGGTATTGAAATCAG 42 1565 1073081 N/A N/A 10692 10711 TTCCAAACGGGCTGGAGAGC 84 1566 1073085 N/A N/A 10804 10823 ATGGACTTTCAGGGCATGAG 64 1567 1073089 N/A N/A 11097 11116 GGAGGGACTGAGGAAACGGA 68 1568 1073093 N/A N/A 11157 11176 GTGTGTATTAGGATCCCATC 24 1569 1073097 N/A N/A 11193 11212 AGCAAGGCCCCCGAGTTTGA 81 1570 1073101 N/A N/A 11246 11265 GCTAGAGTTGGAAGTGAAAG 67 1571 1073105 N/A N/A 11368 11387 ATGAGCTCCCACTGTGGTTG 72 1572 1073109 N/A N/A 11427 11446 GATAACACTGGGAAAGCATT 91 1573 1073113 N/A N/A 11514 11533 AACCTTGGGAAGTCCCCGAC 85 1574 1073117 N/A N/A 11563 11582 ACAGTTAGGAGTTCACACAG 75 1575

TABLE 22 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  5 517 1103152 41 60 3489 3508 CTGGCTCTGCTCGCTCCTGG 91 1576 1103168 272 291 3720 3739 CTCCATCATCTCTGCCCGCT 106  1577 1103184 565 584 4950 4969 CTATAGGCAGCCAGGTTGTT  69* 1578 1103200 760 779 5646 5665 ATCTCTTTCAGGGCTGCGGT 65 1579 1103216 1062 1081 7634 7653 TGAGGCTCTGCCCCTCTTCC 111  1580 1103232 1335 1354 11624 11643 CCTTGTGCTCCTGCTTGGAC 88 1581 1103248 1527 1546 11816 11835 AGGCCTGATACTGACGGAGC 79 1582 1103264 1669 1688 11958 11977 GTAGGTGCCCCCCGCCCTCC 90 1583 1103280 1733 1752 12022 12041 AGAACCTCCATCTCTGGCAA 62 1584 1103296 1860 1879 12149 12168 CCAAAAGACAAAACAAGCCT 75 1585 1103312 1886 1905 12175 12194 CATAGGGATATCCCACCTCA 111  1586 1103328 2094 2113 12383 12402 GTCATCGCTCAGGAGGTCCT 69 1587 1103344 2231 2250 12520 12539 CTATCCCTCCCAGCACCTCA 109  1588 1103360 2427 2446 12716 12735 AGGATGAGTCACTTCCTTAA 73 1589 1103376 2484 2503 12773 12792 GTCATGCCCTGCCCCCATGG 90 1590 1103392 2512 2531 12801 12820 AGGAAGAGGCCTTTAGAAAT 68 1591 1103408 2672 2691 12961 12980 GTGTGTGAGTAAGAAGGGAC 55 1592 1103424 2724 2743 13013 13032 TCAGTTTTACAATTGTAAAA 83 1593 1103440 2899 2918 13188 13207 GGAGAACCCTGAAGTGGGCC 76 1594 1103472 N/A N/A 8787 8806 GTATTGTGAGGCTTTTGAGA 43 1595 1103488 N/A N/A 8899 8918 GAGGCTCACTCCCTGTCAAG 60 1596 1103504 N/A N/A 9045 9064 AACAAGCTCTGCCAGTTTAA 66 1597 1103520 N/A N/A 9234 9253 TGAGTCAGCACTGAGCTGAG 95 1598 1103536 N/A N/A 9270 9289 CAAGAGCTGCGGTCCTGAGG 59 1599 1103552 N/A N/A 9310 9329 TTTAACATTAAGAGCAGGGA 63 1600 1103568 N/A N/A 8532 8551 CTGGTATGATAGGCTCTGGC 86 1601 1103584 N/A N/A 8650 8669 CAGACAGGGCAGATGTCAAG 92 1602 1103600 N/A N/A 3999 4018 CCCCTTCTGCTCACAAGGCC 121  1603 1103616 N/A N/A 4160 4179 CATGCGGGCATCAGATCCCC 89 1604 1103632 N/A N/A 4275 4294 TCGCTGCCCACAGTCACAAA 104  1605 1103648 N/A N/A 4407 4426 GTGCTTTGCGCCCAGACCTG 105  1606 1103664 N/A N/A 4496 4515 GAAATGAATTTTATTATGAC 106  1607 1103680 N/A N/A 4640 4659 TCCACAAGCATACACTCACT 81 1608 1103696 N/A N/A 4837 4856 TGCCTCAGTCTCCCTTGAGG 107  1609 1103712 N/A N/A 5100 5119 CCATTCCTCAGCCTTGCCTT 98 1610 1103728 N/A N/A 5346 5365 GTCTCTCTCAGTCTCAGCTT 78 1611 1103744 N/A N/A 5413 5432 GTCTTTCTGTTTGTCTTTCA 57 1612 1103760 N/A N/A 5487 5506 CCATTGTGTCCTCTTCTGCC 102  1613 1103776 N/A N/A 5776 5795 TTCTCTTGTACAGAGCAAGA 79 1614 1103792 N/A N/A 5860 5879 CTGGGCAAGCCATCTCACTT 86 1615 1103808 N/A N/A 5941 5960 CAGTATCAGCTACTACTAAT 80 1616 1103824 N/A N/A 5978 5997 AAGGTAATCCATGAAAAGCA 84 1617 1103840 N/A N/A 6024 6043 GACTTCGGCTCTCTCATCTG 82 1618 1103856 N/A N/A 6204 6223 CCAGGTCAGACACCTCTCTG 81 1619 1103872 N/A N/A 6267 6286 AAGCTCTGAGCTGTGGTGTT 33 1620 1103888 N/A N/A 6315 6334 CCCAAATGCCCCCTCTACAG 93 1621 1103904 N/A N/A 6414 6433 CCCTGCCTCTCTGTGCTTTT 92 1622 1103920 N/A N/A 6517 6536 AGGTCCTCCCAGCCCCATCG 108  1623 1103936 N/A N/A 6947 6966 TCCACACTGACAGCTGCATC 92 1624 1103952 N/A N/A 7030 7049 TAGCGGGCTGCCAGACCTCA 102  1625 1103968 N/A N/A 7326 7345 CCCTGGCCGCGCTCACCGTG 96 1626 1103984 N/A N/A 7421 7440 TTCAGGCCCCGCCCTCGACC 81 1627 1104000 N/A N/A 7814 7833 AGGGAGCAAGATGAGCTCTA 120  1628 1104016 N/A N/A 7940 7959 GAATCCATCCATCCATTCAG 86 1629 1104032 N/A N/A 8038 8057 GGTGAGGAGTCCAATCTTGG 76 1630 1104048 N/A N/A 8080 8099 AAGTGGTGAAGAAAGTTCCA 108  1631 1104064 N/A N/A 8251 8270 CATCATGACAACTTGAACGC 98 1632 1104080 N/A N/A 9364 9383 CCTCCCCCCGCCCCGCCCGA 111  1633 1104096 N/A N/A 9414 9433 GCTAAGAATCATTTCAGGGC 40 1634 1104112 N/A N/A 9455 9474 CTAAATATTCTAGTCCAGAA 97 1635 1104128 N/A N/A 9509 9528 TGGTTAGCCTTTCTGATGCT 84 1636 1104144 N/A N/A 9535 9554 GGCAGTATTACCTCTACTAG 28 1637 1104160 N/A N/A 9559 9578 GTGACCTGTGACTATCTAGG 47 1638 1104176 N/A N/A 9579 9598 TGCCATTTATCTGTGCTTTA 50 1639 1104192 N/A N/A 9665 9684 TAAAGGCTGTTAAACATGTG 51 1640 1104208 N/A N/A 9785 9804 ACCAGCCACATGACTCTGGG 83 1641 1104224 N/A N/A 10558 10577 CACGGTATTGAAATCAGGAG 47 1642 1104240 N/A N/A 10654 10673 CTGCTGCCAGAGTCCTGGCT 88 1643 1104256 N/A N/A 10753 10772 CACCCCCCTCCCCATCATGA 89 1644 1104272 N/A N/A 10818 10837 AGAGGAGGCCTCTCATGGAC 96 1645 1104288 N/A N/A 11083 11102 AACGGAATTACATTCAGTTT 81 1646 1104304 N/A N/A 11152 11171 TATTAGGATCCCATCTAGTG 103  1647 1104320 N/A N/A 11230 11249 AAAGCTACTAACTTTAATTC 81 1648 1104336 N/A N/A 11288 11307 CAAGCTGGAAATGGAAAGCC 83 1649 1104352 N/A N/A 11390 11409 AGTGGTAAGCTGCTGGAGTA 85 1650 1104368 N/A N/A 11499 11518 CCGACTTCCCAGGTCTTACT 78 1651

TABLE 23 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  6 517 1103150 20 39 3468 3487 ATGCGAGGGCTTTATGAAGG 82 1652 1103166 264 283 3712 3731 TCTCTGCCCGCTCACTGGCC 126  1653 1103182 549 568 4934 4953 TGTTCTCGGCTTCCAGCCTC  40* 1654 1103198 758 777 5644 5663 CTCTTTCAGGGCTGCGGTGA 35 1655 1103214 1049 1068 7621 7640 CTCTTCCTCCAGCCGCGCCA 70 1656 1103230 1327 1346 11616 11635 TCCTGCTTGGACTCCTTAAT 69 1657 1103246 1501 1520 11790 11809 CAGCAAGCTGACCTAGGGAC 39 1658 1103262 1662 1681 11951 11970 CCCCCCGCCCTCCTCCCCTT 91 1659 1103278 1729 1748 12018 12037 CCTCCATCTCTGGCAACAGT 34 1660 1103294 1858 1877 12147 12166 AAAAGACAAAACAAGCCTCT 81 1661 1103310 1884 1903 12173 12192 TAGGGATATCCCACCTCATA 74 1662 1103326 2061 2080 12350 12369 CAGGTGACTGCCCCAGGTGG 73 1663 1103342 2228 2247 12517 12536 TCCCTCCCAGCACCTCATCC 80 1664 1103358 2424 2443 12713 12732 ATGAGTCACTTCCTTAATTC 58 1665 1103374 2467 2486 12756 12775 TGGATACATCCCCTTTCTCT 59 1666 1103390 2510 2529 12799 12818 GAAGAGGCCTTTAGAAATGG 76 1667 1103406 2668 2687 12957 12976 GTGAGTAAGAAGGGACCGCA 82 1668 1103422 2717 2736 13006 13025 TACAATTGTAAAATAGGGCA 90 1669 1103438 2849 2868 13138 13157 ACCAGTCTGCTCACCAGTCT 74 1670 1103470 N/A N/A 8769 8788 GATATCTTGTGACCTTGTGA 34 1671 1103486 N/A N/A 8897 8916 GGCTCACTCCCTGTCAAGCT 100  1672 1103502 N/A N/A 9043 9062 CAAGCTCTGCCAGTTTAATG 30 1673 1103518 N/A N/A 9224 9243 CTGAGCTGAGCGATGGAGCC 71 1674 1103534 N/A N/A 9263 9282 TGCGGTCCTGAGGGAAGAAT 76 1675 1103550 N/A N/A 9308 9327 TAACATTAAGAGCAGGGAAC 77 1676 1103566 N/A N/A 8500 8519 AGTGTCACGAAGGCCCCCAG 49 1677 1103582 N/A N/A 8584 8603 GGACCAGGGCCTAGCAGGAC 69 1678 1103598 N/A N/A 3993 4012 CTGCTCACAAGGCCCCCCTT 92 1679 1103614 N/A N/A 4049 4068 GGTTCGGCCCCTCCCTGAGA 69 1680 1103630 N/A N/A 4250 4269 GCCATGAATGAAACACAGGG 66 1681 1103646 N/A N/A 4391 4410 CCTGCCTGCTCTTTCCCTCA 76 1682 1103662 N/A N/A 4490 4509 AATTTTATTATGACCACCGC 110  1683 1103678 N/A N/A 4635 4654 AAGCATACACTCACTGTTGC 78 1684 1103694 N/A N/A 4817 4836 CAGCTGTCACAGAGACCACC 100  1685 1103710 N/A N/A 5097 5116 TTCCTCAGCCTTGCCTTACC 78 1686 1103726 N/A N/A 5305 5324 TCTCAGTTGCAATCTCTGTG 87 1687 1103742 N/A N/A 5398 5417 TTTCATTTCCTGTCTCTACC 89 1688 1103758 N/A N/A 5481 5500 TGTCCTCTTCTGCCTGCCCC 94 1689 1103774 N/A N/A 5726 5745 CAGGCAGGGCTACCTTGGAG 87 1690 1103790 N/A N/A 5849 5868 ATCTCACTTCTCTGGTGAAA 95 1691 1103806 N/A N/A 5923 5942 ATAATAGCAATAGTAGCAGT 103  1692 1103822 N/A N/A 5974 5993 TAATCCATGAAAAGCACAGG 84 1693 1103838 N/A N/A 6020 6039 TCGGCTCTCTCATCTGTCAA 53 1694 1103854 N/A N/A 6199 6218 TCAGACACCTCTCTGTGTCC 86 1695 1103870 N/A N/A 6257 6276 CTGTGGTGTTCTCTACGGGC 78 1696 1103886 N/A N/A 6313 6332 CAAATGCCCCCTCTACAGTG 105  1697 1103902 N/A N/A 6408 6427 CTCTCTGTGCTTTTCTGCCT 71 1698 1103918 N/A N/A 6501 6520 ATCGGGCCCTCACCCTGCTC 107  1699 1103934 N/A N/A 6904 6923 TAGGCGAGCGGAGGCCTGGG 90 1700 1103950 N/A N/A 7017 7036 GACCTCAGCACCTAGCACAA 82 1701 1103966 N/A N/A 7314 7333 TCACCGTGCCGCGCAGAGAC 101  1702 1103982 N/A N/A 7416 7435 GCCCCGCCCTCGACCCAGGT 88 1703 1103998 N/A N/A 7805 7824 GATGAGCTCTACCGTGAGGC 71 1704 1104014 N/A N/A 7922 7941 AGTCATCAAACATCTAGTGA 46 1705 1104030 N/A N/A 8035 8054 GAGGAGTCCAATCTTGGCTG 67 1706 1104046 N/A N/A 8075 8094 GTGAAGAAAGTTCCAAGGAG 93 1707 1104062 N/A N/A 8228 8247 TTTCCTTGCCAGGAAAGTCT 81 1708 1104078 N/A N/A 9359 9378 CCCCGCCCCGCCCGAGAGAG 36 1709 1104094 N/A N/A 9412 9431 TAAGAATCATTTCAGGGCCA 71 1710 1104110 N/A N/A 9452 9471 AATATTCTAGTCCAGAAGAA 103  1711 1104126 N/A N/A 9506 9525 TTAGCCTTTCTGATGCTGAA 58 1712 1104142 N/A N/A 9530 9549 TATTACCTCTACTAGTCAGC 65 1713 1104158 N/A N/A 9557 9576 GACCTGTGACTATCTAGGAT 18 1714 1104174 N/A N/A 9576 9595 CATTTATCTGTGCTTTAGTG 37 1715 1104190 N/A N/A 9660 9679 GCTGTTAAACATGTGGCACA 74 1716 1104206 N/A N/A 9706 9725 CCTACTTCTCTAGGTGGGAG 65 1717 1104222 N/A N/A 10556 10575 CGGTATTGAAATCAGGAGAC 47 1718 1104238 N/A N/A 10610 10629 TTTCAGCCCCTCTGCAAGCC 68 1719 1104254 N/A N/A 10747 10766 CCTCCCCATCATGAGTATGA 92 1720 1104270 N/A N/A 10816 10835 AGGAGGCCTCTCATGGACTT 93 1721 1104286 N/A N/A 11078 11097 AATTACATTCAGTTTCCTTG 88 1722 1104302 N/A N/A 11150 11169 TTAGGATCCCATCTAGTGGC 60 1723 1104318 N/A N/A 11225 11244 TACTAACTTTAATTCTCTTT 49 1724 1104334 N/A N/A 11280 11299 AAATGGAAAGCCCTCCCCAT 98 1725 1104350 N/A N/A 11388 11407 TGGTAAGCTGCTGGAGTAAG 55 1726 1104366 N/A N/A 11494 11513 TTCCCAGGTCTTACTTTTCT 76 1727

TABLE 24 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  6 517 1103151 39 58 3487 3506 GGCTCTGCTCGCTCCTGGGA 108  1728 1103167 269 288 3717 3736 CATCATCTCTGCCCGCTCAC 93 1729 1103183 553 572 4938 4957 AGGTTGTTCTCGGCTTCCAG  55* 1730 1103199 759 778 5645 5664 TCTCTTTCAGGGCTGCGGTG 54 1731 1103215 1050 1069 7622 7641 CCTCTTCCTCCAGCCGCGCC 64 1732 1103231 1334 1353 11623 11642 CTTGTGCTCCTGCTTGGACT 53 1733 1103247 1518 1537 11807 11826 ACTGACGGAGCCTAGGGCAG 61 1734 1103263 1667 1686 11956 11975 AGGTGCCCCCCGCCCTCCTC 98 1735 1103279 1730 1749 12019 12038 ACCTCCATCTCTGGCAACAG 25 1736 1103295 1859 1878 12148 12167 CAAAAGACAAAACAAGCCTC 73 1737 1103311 1885 1904 12174 12193 ATAGGGATATCCCACCTCAT 75 1738 1103327 2086 2105 12375 12394 TCAGGAGGTCCTTCTGGGAT 67 1739 1103343 2230 2249 12519 12538 TATCCCTCCCAGCACCTCAT 63 1740 1103359 2425 2444 12714 12733 GATGAGTCACTTCCTTAATT 35 1741 1103375 2483 2502 12772 12791 TCATGCCCTGCCCCCATGGA 69 1742 1103391 2511 2530 12800 12819 GGAAGAGGCCTTTAGAAATG 74 1743 1103407 2670 2689 12959 12978 GTGTGAGTAAGAAGGGACCG 46 1744 1103423 2722 2741 13011 13030 AGTTTTACAATTGTAAAATA 88 1745 1103439 2868 2887 13157 13176 ATCTCTGGGCACAGATCCCA 93 1746 1103471 N/A N/A 8770 8789 AGATATCTTGTGACCTTGTG 35 1747 1103487 N/A N/A 8898 8917 AGGCTCACTCCCTGTCAAGC 62 1748 1103503 N/A N/A 9044 9063 ACAAGCTCTGCCAGTTTAAT 51 1749 1103519 N/A N/A 9225 9244 ACTGAGCTGAGCGATGGAGC 65 1750 1103535 N/A N/A 9266 9285 AGCTGCGGTCCTGAGGGAAG 59 1751 1103551 N/A N/A 9309 9328 TTAACATTAAGAGCAGGGAA 68 1752 1103567 N/A N/A 8530 8549 GGTATGATAGGCTCTGGCTA 21 1753 1103583 N/A N/A 8649 8668 AGACAGGGCAGATGTCAAGC 90 1754 1103599 N/A N/A 3994 4013 TCTGCTCACAAGGCCCCCCT 56 1755 1103615 N/A N/A 4053 4072 GGGAGGTTCGGCCCCTCCCT 94 1756 1103631 N/A N/A 4251 4270 AGCCATGAATGAAACACAGG 115  1757 1103647 N/A N/A 4401 4420 TGCGCCCAGACCTGCCTGCT 75 1758 1103663 N/A N/A 4495 4514 AAATGAATTTTATTATGACC 80 1759 1103679 N/A N/A 4636 4655 CAAGCATACACTCACTGTTG 69 1760 1103695 N/A N/A 4820 4839 AGGCAGCTGTCACAGAGACC 95 1761 1103711 N/A N/A 5099 5118 CATTCCTCAGCCTTGCCTTA 95 1762 1103727 N/A N/A 5311 5330 TCCCTCTCTCAGTTGCAATC 79 1763 1103743 N/A N/A 5412 5431 TCTTTCTGTTTGTCTTTCAT 71 1764 1103759 N/A N/A 5482 5501 GTGTCCTCTTCTGCCTGCCC 59 1765 1103775 N/A N/A 5775 5794 TCTCTTGTACAGAGCAAGAA 110  1766 1103791 N/A N/A 5853 5872 AGCCATCTCACTTCTCTGGT 67 1767 1103807 N/A N/A 5930 5949 ACTACTAATAATAGCAATAG 82 1768 1103823 N/A N/A 5975 5994 GTAATCCATGAAAAGCACAG 97 1769 1103839 N/A N/A 6022 6041 CTTCGGCTCTCTCATCTGTC 76 1770 1103855 N/A N/A 6203 6222 CAGGTCAGACACCTCTCTGT 76 1771 1103871 N/A N/A 6265 6284 GCTCTGAGCTGTGGTGTTCT 88 1772 1103887 N/A N/A 6314 6333 CCAAATGCCCCCTCTACAGT 92 1773 1103903 N/A N/A 6409 6428 CCTCTCTGTGCTTTTCTGCC 60 1774 1103919 N/A N/A 6516 6535 GGTCCTCCCAGCCCCATCGG 79 1775 1103935 N/A N/A 6906 6925 CTTAGGCGAGCGGAGGCCTG 85 1776 1103951 N/A N/A 7021 7040 GCCAGACCTCAGCACCTAGC 65 1777 1103967 N/A N/A 7316 7335 GCTCACCGTGCCGCGCAGAG 76 1778 1103983 N/A N/A 7420 7439 TCAGGCCCCGCCCTCGACCC 88 1779 1103999 N/A N/A 7806 7825 AGATGAGCTCTACCGTGAGG 66 1780 1104015 N/A N/A 7930 7949 ATCCATTCAGTCATCAAACA 90 1781 1104031 N/A N/A 8037 8056 GTGAGGAGTCCAATCTTGGC 77 1782 1104047 N/A N/A 8076 8095 GGTGAAGAAAGTTCCAAGGA 59 1783 1104063 N/A N/A 8229 8248 TTTTCCTTGCCAGGAAAGTC 76 1784 1104079 N/A N/A 9360 9379 CCCCCGCCCCGCCCGAGAGA 103  1785 1104095 N/A N/A 9413 9432 CTAAGAATCATTTCAGGGCC 65 1786 1104111 N/A N/A 9454 9473 TAAATATTCTAGTCCAGAAG 87 1787 1104127 N/A N/A 9508 9527 GGTTAGCCTTTCTGATGCTG 30 1788 1104143 N/A N/A 9531 9550 GTATTACCTCTACTAGTCAG 56 1789 1104159 N/A N/A 9558 9577 TGACCTGTGACTATCTAGGA 29 1790 1104175 N/A N/A 9578 9597 GCCATTTATCTGTGCTTTAG 10 1791 1104191 N/A N/A 9664 9683 AAAGGCTGTTAAACATGTGG 39 1792 1104207 N/A N/A 9707 9726 GCCTACTTCTCTAGGTGGGA 69 1793 1104223 N/A N/A 10557 10576 ACGGTATTGAAATCAGGAGA 55 1794 1104239 N/A N/A 10639 10658 TGGCTGCTCTGTCTTCTGGC 52 1795 1104255 N/A N/A 10748 10767 CCCTCCCCATCATGAGTATG 79 1796 1104271 N/A N/A 10817 10836 GAGGAGGCCTCTCATGGACT 95 1797 1104287 N/A N/A 11082 11101 ACGGAATTACATTCAGTTTC 77 1798 1104303 N/A N/A 11151 11170 ATTAGGATCCCATCTAGTGG 68 1799 1104319 N/A N/A 11229 11248 AAGCTACTAACTTTAATTCT 92 1800 1104335 N/A N/A 11281 11300 GAAATGGAAAGCCCTCCCCA 113  1801 1104351 N/A N/A 11389 11408 GTGGTAAGCTGCTGGAGTAA 60 1802 1104367 N/A N/A 11498 11517 CGACTTCCCAGGTCTTACTT 74 1803

TABLE 25 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  7 517 1103153 44 63 3492 3511 GCTCTGGCTCTGCTCGCTCC 83 1804 1103169 354 373 3802 3821 GGTTCAGCTCAGCAGCCAGC 85 1805 1103185 566 585 4951 4970 TCTATAGGCAGCCAGGTTGT  73* 1806 1103201 762 781 5648 5667 GGATCTCTTTCAGGGCTGCG 90 1807 1103217 1185 1204 N/A N/A GAATGGTGATCCGGTTCTCC 23 1808 1103233 1350 1369 11639 11658 TGCCTCACATCACATCCTTG 98 1809 1103249 1528 1547 11817 11836 CAGGCCTGATACTGACGGAG 103  1810 1103265 1670 1689 11959 11978 AGTAGGTGCCCCCCGCCCTC 74 1811 1103281 1750 1769 12039 12058 TTCCCAGATACTCCGAGAGA 65 1812 1103297 1861 1880 12150 12169 ACCAAAAGACAAAACAAGCC 113  1813 1103313 1887 1906 12176 12195 GCATAGGGATATCCCACCTC 95 1814 1103329 2097 2116 12386 12405 TGAGTCATCGCTCAGGAGGT 47 1815 1103345 2331 2350 12620 12639 TCCTCCTCCATCTCTACCAG 44 1816 1103361 2431 2450 12720 12739 CAAGAGGATGAGTCACTTCC 49 1817 1103377 2485 2504 12774 12793 AGTCATGCCCTGCCCCCATG 46 1818 1103393 2516 2535 12805 12824 AGCAAGGAAGAGGCCTTTAG 68 1819 1103409 2673 2692 12962 12981 TGTGTGTGAGTAAGAAGGGA 30 1820 1103425 2725 2744 13014 13033 CTCAGTTTTACAATTGTAAA 71 1821 1103441 2905 2924 13194 13213 GGGAGAGGAGAACCCTGAAG 98 1822 1103473 N/A N/A 8813 8832 CTGGTGAGCCTGTATTGGTA 68 1823 1103489 N/A N/A 8975 8994 AAAATGACGCAGTCCAGGCC 108  1824 1103505 N/A N/A 9046 9065 TAACAAGCTCTGCCAGTTTA 92 1825 1103521 N/A N/A 9238 9257 GAACTGAGTCAGCACTGAGC 124  1826 1103537 N/A N/A 9279 9298 TATTCACTGCAAGAGCTGCG 111  1827 1103553 N/A N/A 9313 9332 ATATTTAACATTAAGAGCAG 84 1828 1103569 N/A N/A 8533 8552 CCTGGTATGATAGGCTCTGG 56 1829 1103585 N/A N/A 8655 8674 ATCTGCAGACAGGGCAGATG 113  1830 1103601 N/A N/A 4001 4020 AGCCCCTTCTGCTCACAAGG 74 1831 1103617 N/A N/A 4172 4191 CCTGGCAGAAGGCATGCGGG 120  1832 1103633 N/A N/A 4295 4314 CAGGCTCAGAATAGGTGAGC 80 1833 1103649 N/A N/A 4410 4429 CCTGTGCTTTGCGCCCAGAC 92 1834 1103665 N/A N/A 4505 4524 CCACCTTTTGAAATGAATTT 63 1835 1103681 N/A N/A 4641 4660 ATCCACAAGCATACACTCAC 62 1836 1103697 N/A N/A 4845 4864 TGAATACCTGCCTCAGTCTC 68 1837 1103713 N/A N/A 5101 5120 CCCATTCCTCAGCCTTGCCT 95 1838 1103729 N/A N/A 5351 5370 TGAGTGTCTCTCTCAGTCTC 132  1839 1103745 N/A N/A 5420 5439 CCTTAGTGTCTTTCTGTTTG 69 1840 1103761 N/A N/A 5489 5508 CGCCATTGTGTCCTCTTCTG 86 1841 1103777 N/A N/A 5778 5797 CATTCTCTTGTACAGAGCAA 105  1842 1103793 N/A N/A 5861 5880 CCTGGGCAAGCCATCTCACT 119  1843 1103809 N/A N/A 5943 5962 AACAGTATCAGCTACTACTA 80 1844 1103825 N/A N/A 5979 5998 TAAGGTAATCCATGAAAAGC 101  1845 1103841 N/A N/A 6108 6127 CCCAAGTGAGATGTGCTAGA 118  1846 1103857 N/A N/A 6205 6224 TCCAGGTCAGACACCTCTCT 97 1847 1103873 N/A N/A 6268 6287 TAAGCTCTGAGCTGTGGTGT 51 1848 1103889 N/A N/A 6316 6335 GCCCAAATGCCCCCTCTACA 73 1849 1103905 N/A N/A 6415 6434 GCCCTGCCTCTCTGTGCTTT 64 1850 1103921 N/A N/A 6522 6541 CAGGGAGGTCCTCCCAGCCC 101  1851 1103937 N/A N/A 6948 6967 TTCCACACTGACAGCTGCAT 76 1852 1103953 N/A N/A 7077 7096 TGGCTGGGACTTTTCCCAAC 113  1853 1103969 N/A N/A 7330 7349 CCTGCCCTGGCCGCGCTCAC 87 1854 1103985 N/A N/A 7423 7442 CGTTCAGGCCCCGCCCTCGA 78 1855 1104001 N/A N/A 7815 7834 AAGGGAGCAAGATGAGCTCT 77 1856 1104017 N/A N/A 7956 7975 CTAATCAATATTGGTTGAAT 87 1857 1104033 N/A N/A 8039 8058 AGGTGAGGAGTCCAATCTTG 82 1858 1104049 N/A N/A 8082 8101 GAAAGTGGTGAAGAAAGTTC 109  1859 1104065 N/A N/A 8252 8271 CCATCATGACAACTTGAACG 110  1860 1104081 N/A N/A 9377 9396 AATTTAGCTCCCCCCTCCCC 83 1861 1104097 N/A N/A 9417 9436 TTAGCTAAGAATCATTTCAG 125  1862 1104113 N/A N/A 9456 9475 CCTAAATATTCTAGTCCAGA 47 1863 1104129 N/A N/A 9511 9530 CCTGGTTAGCCTTTCTGATG 47 1864 1104145 N/A N/A 9537 9556 TTGGCAGTATTACCTCTACT 19 1865 1104161 N/A N/A 9561 9580 TAGTGACCTGTGACTATCTA 47 1866 1104177 N/A N/A 9580 9599 CTGCCATTTATCTGTGCTTT 52 1867 1104193 N/A N/A 9672 9691 ACTAAAATAAAGGCTGTTAA 67 1868 1104209 N/A N/A 9786 9805 AACCAGCCACATGACTCTGG 91 1869 1104225 N/A N/A 10559 10578 GCACGGTATTGAAATCAGGA 30 1870 1104241 N/A N/A 10655 10674 GCTGCTGCCAGAGTCCTGGC 69 1871 1104257 N/A N/A 10761 10780 CCCCATCGCACCCCCCTCCC 103  1872 1104273 N/A N/A 10843 10862 CAGGCTGGTTTCTGCAGATG 92 1873 1104289 N/A N/A 11084 11103 AAACGGAATTACATTCAGTT 67 1874 1104305 N/A N/A 11153 11172 GTATTAGGATCCCATCTAGT 58 1875 1104321 N/A N/A 11231 11250 GAAAGCTACTAACTTTAATT 60 1876 1104337 N/A N/A 11308 11327 CCCTGGTAGTTTCCTTTTAC 55 1877 1104353 N/A N/A 11392 11411 CGAGTGGTAAGCTGCTGGAG 69 1878 1104369 N/A N/A 11512 11531 CCTTGGGAAGTCCCCGACTT 78 1879

TABLE 26 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  6 517 1103154 109 128 3557 3576 ATCATCTCCCCTGAGGAGAC 139  1880 1103170 376 395 3824 3843 GTGGGCTCCTTGGCCCGCAG 89 1881 1103186 576 595 N/A N/A CTGCTTCCTGTCTATAGGCA  30* 1882 1103202 763 782 5649 5668 CGGATCTCTTTCAGGGCTGC 38 1883 1103218 1202 1221 8351 8370 GGAGAAGGTCTGCACGGGAA 43 1884 1103234 1355 1374 11644 11663 GGTCCTGCCTCACATCACAT 87 1885 1103250 1532 1551 11821 11840 CTGGCAGGCCTGATACTGAC 93 1886 1103266 1679 1698 11968 11987 GGGCGATGTAGTAGGTGCCC 100  1887 1103282 1762 1781 12051 12070 TCAAAGGCACAGTTCCCAGA 89 1888 1103298 1862 1881 12151 12170 AACCAAAAGACAAAACAAGC 80 1889 1103314 1889 1908 12178 12197 CAGCATAGGGATATCCCACC 86 1890 1103330 2098 2117 12387 12406 TTGAGTCATCGCTCAGGAGG 49 1891 1103346 2332 2351 12621 12640 CTCCTCCTCCATCTCTACCA 81 1892 1103362 2436 2455 12725 12744 ATCTTCAAGAGGATGAGTCA 116  1893 1103378 2487 2506 12776 12795 AAAGTCATGCCCTGCCCCCA 74 1894 1103394 2526 2545 12815 12834 GGTATGACACAGCAAGGAAG 59 1895 1103410 2674 2693 12963 12982 TTGTGTGTGAGTAAGAAGGG 52 1896 1103426 2730 2749 13019 13038 CGTGCCTCAGTTTTACAATT 73 1897 1103442 2963 2982 13252 13271 TGGAGGGAAAGGACACCAAG 79 1898 1103458 N/A N/A 8740 8759 TCTTTGGTGCTTTTGCCCCC 50 1899 1103474 N/A N/A 8814 8833 TCTGGTGAGCCTGTATTGGT 66 1900 1103490 N/A N/A 8978 8997 GGGAAAATGACGCAGTCCAG 80 1901 1103506 N/A N/A 9076 9095 GCGCACCCAAGGACTCACCA 115  1902 1103522 N/A N/A 9240 9259 CTGAACTGAGTCAGCACTGA 90 1903 1103538 N/A N/A 9285 9304 AAACTTTATTCACTGCAAGA 57 1904 1103570 N/A N/A 8537 8556 GTACCCTGGTATGATAGGCT 22 1905 1103586 N/A N/A 8684 8703 ACACTCAGAAGGGCAGTGCT 115  1906 1103602 N/A N/A 4002 4021 CAGCCCCTTCTGCTCACAAG 77 1907 1103618 N/A N/A 4181 4200 CCCTGGACTCCTGGCAGAAG 98 1908 1103634 N/A N/A 4336 4355 CATCAACCTTCTCCGCTTCC 75 1909 1103650 N/A N/A 4411 4430 ACCTGTGCTTTGCGCCCAGA 92 1910 1103666 N/A N/A 4566 4585 TGCATGTCCTGTCAGCTCAG 122  1911 1103682 N/A N/A 4645 4664 GCGCATCCACAAGCATACAC 113  1912 1103698 N/A N/A 4846 4865 TTGAATACCTGCCTCAGTCT 104  1913 1103714 N/A N/A 5140 5159 TGAGGAGTAGAGGGCCACTG 64 1914 1103730 N/A N/A 5353 5372 TCTGAGTGTCTCTCTCAGTC 135  1915 1103746 N/A N/A 5422 5441 TCCCTTAGTGTCTTTCTGTT 101  1916 1103762 N/A N/A 5494 5513 CCCCACGCCATTGTGTCCTC 85 1917 1103778 N/A N/A 5780 5799 CCCATTCTCTTGTACAGAGC 72 1918 1103794 N/A N/A 5862 5881 CCCTGGGCAAGCCATCTCAC 80 1919 1103810 N/A N/A 5948 5967 AACAGAACAGTATCAGCTAC 82 1920 1103826 N/A N/A 5980 5999 GTAAGGTAATCCATGAAAAG 73 1921 1103842 N/A N/A 6112 6131 GGCTCCCAAGTGAGATGTGC 118  1922 1103858 N/A N/A 6207 6226 CTTCCAGGTCAGACACCTCT 75 1923 1103874 N/A N/A 6269 6288 ATAAGCTCTGAGCTGTGGTG 50 1924 1103890 N/A N/A 6317 6336 AGCCCAAATGCCCCCTCTAC 77 1925 1103906 N/A N/A 6449 6468 CAGCTAGGTGCCCTGGCTAG 131  1926 1103922 N/A N/A 6531 6550 CCCTGCCTGCAGGGAGGTCC 69 1927 1103938 N/A N/A 6970 6989 CAGTGCCACAGAATCCAGAA 94 1928 1103954 N/A N/A 7104 7123 CCCCAGTTAACCCCAGGACG 130  1929 1103970 N/A N/A 7337 7356 TCCCCGTCCTGCCCTGGCCG 65 1930 1103986 N/A N/A 7454 7473 GCCCCAGGCCCCGCCTCTAG 174  1931 1104002 N/A N/A 7824 7843 CAGTCCCTGAAGGGAGCAAG 113  1932 1104018 N/A N/A 7958 7977 GCCTAATCAATATTGGTTGA 71 1933 1104034 N/A N/A 8041 8060 TGAGGTGAGGAGTCCAATCT 69 1934 1104050 N/A N/A 8107 8126 CTGAAGGAAGATGGAAAAGG 78 1935 1104066 N/A N/A 8254 8273 GGCCATCATGACAACTTGAA 105  1936 1104082 N/A N/A 9378 9397 TAATTTAGCTCCCCCCTCCC 106  1937 1104098 N/A N/A 9418 9437 CTTAGCTAAGAATCATTTCA 96 1938 1104114 N/A N/A 9457 9476 TCCTAAATATTCTAGTCCAG 63 1939 1104130 N/A N/A 9513 9532 AGCCTGGTTAGCCTTTCTGA 68 1940 1104146 N/A N/A 9538 9557 TTTGGCAGTATTACCTCTAC 73 1941 1104162 N/A N/A 9562 9581 TTAGTGACCTGTGACTATCT 92 1942 1104178 N/A N/A 9582 9601 CTCTGCCATTTATCTGTGCT 48 1943 1104194 N/A N/A 9675 9694 TTAACTAAAATAAAGGCTGT 132  1944 1104210 N/A N/A 9810 9829 TCAAGAAGTCCCAACTTAGC 74 1945 1104226 N/A N/A 10560 10579 AGCACGGTATTGAAATCAGG 77 1946 1104242 N/A N/A 10656 10675 TGCTGCTGCCAGAGTCCTGG 89 1947 1104258 N/A N/A 10773 10792 CATGCCCGGCTTCCCCATCG 98 1948 1104274 N/A N/A 10923 10942 CTTACCTCTCCATCCCGCAT 100  1949 1104290 N/A N/A 11088 11107 GAGGAAACGGAATTACATTC 95 1950 1104306 N/A N/A 11154 11173 TGTATTAGGATCCCATCTAG 135  1951 1104322 N/A N/A 11232 11251 TGAAAGCTACTAACTTTAAT 75 1952 1104338 N/A N/A 11312 11331 ATTCCCCTGGTAGTTTCCTT 97 1953 1104354 N/A N/A 11393 11412 GCGAGTGGTAAGCTGCTGGA 57 1954 1104370 N/A N/A 11523 11542 AGGCTGTGTAACCTTGGGAA 75 1955

TABLE 27 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  7 517 1103155 110 129 3558 3577 CATCATCTCCCCTGAGGAGA 90 1956 1103171 380 399 3828 3847 CTTGGTGGGCTCCTTGGCCC 103  1957 1103187 577 596 N/A N/A TCTGCTTCCTGTCTATAGGC  33* 1958 1103203 764 783 5650 5669 GCGGATCTCTTTCAGGGCTG 65 1959 1103219 1220 1239 N/A N/A TTCTCGAATCTGCAGGTTGG 111  1960 1103235 1361 1380 11650 11669 CAGGTGGGTCCTGCCTCACA 147  1961 1103251 1533 1552 11822 11841 TCTGGCAGGCCTGATACTGA 134  1962 1103267 1681 1700 11970 11989 GAGGGCGATGTAGTAGGTGC 66 1963 1103283 1768 1787 12057 12076 GGAAACTCAAAGGCACAGTT 72 1964 1103299 1870 1889 12159 12178 CTCATAAAAACCAAAAGACA 128  1965 1103315 1890 1909 12179 12198 GCAGCATAGGGATATCCCAC 89 1966 1103331 2107 2126 12396 12415 CTGAGACACTTGAGTCATCG 45 1967 1103347 2334 2353 12623 12642 GCCTCCTCCTCCATCTCTAC 53 1968 1103363 2440 2459 12729 12748 CAGCATCTTCAAGAGGATGA 109  1969 1103379 2488 2507 12777 12796 CAAAGTCATGCCCTGCCCCC 94 1970 1103395 2527 2546 12816 12835 TGGTATGACACAGCAAGGAA 48 1971 1103411 2676 2695 12965 12984 TTTTGTGTGTGAGTAAGAAG 43 1972 1103427 2750 2769 13039 13058 GCCAGTGTCTTCACTTTGCT 73 1973 1103443 3013 3032 13302 13321 CAGTGCCCTGAAGATTAGCA 68 1974 1103459 N/A N/A 8741 8760 GTCTTTGGTGCTTTTGCCCC 51 1975 1103475 N/A N/A 8815 8834 ATCTGGTGAGCCTGTATTGG 45 1976 1103491 N/A N/A 8979 8998 TGGGAAAATGACGCAGTCCA 61 1977 1103507 N/A N/A 9091 9110 CAGAGCAGCTCCACTGCGCA 127  1978 1103523 N/A N/A 9241 9260 TCTGAACTGAGTCAGCACTG 84 1979 1103539 N/A N/A 9286 9305 AAAACTTTATTCACTGCAAG 64 1980 1103555 N/A N/A 8369 8388 ACCTCGAATCTGCAGGTTGG 130  1981 1103571 N/A N/A 8538 8557 AGTACCCTGGTATGATAGGC 33 1982 1103587 N/A N/A 8714 8733 GACAAGCAGTTAAAAAAACA 62 1983 1103603 N/A N/A 4003 4022 TCAGCCCCTTCTGCTCACAA 58 1984 1103619 N/A N/A 4210 4229 CTTTCCCCAGTAGGGAGGTG 86 1985 1103635 N/A N/A 4343 4362 TCATGGACATCAACCTTCTC 87 1986 1103651 N/A N/A 4414 4433 GTCACCTGTGCTTTGCGCCC 107  1987 1103667 N/A N/A 4568 4587 CATGCATGTCCTGTCAGCTC 72 1988 1103683 N/A N/A 4646 4665 GGCGCATCCACAAGCATACA 112  1989 1103699 N/A N/A 4855 4874 TGAGGACACTTGAATACCTG 67 1990 1103715 N/A N/A 5163 5182 GCTTCCTGGAGTGGCAGGAG 109* 1991 1103731 N/A N/A 5362 5381 TCTCCTCTCTCTGAGTGTCT 69 1992 1103747 N/A N/A 5424 5443 TCTCCCTTAGTGTCTTTCTG 65 1993 1103763 N/A N/A 5499 5518 GGGTTCCCCACGCCATTGTG 109  1994 1103779 N/A N/A 5782 5801 CCCCCATTCTCTTGTACAGA 136  1995 1103795 N/A N/A 5881 5900 AGCAATAGTGCCTGTGTGAC 69 1996 1103811 N/A N/A 5949 5968 GAACAGAACAGTATCAGCTA 91 1997 1103827 N/A N/A 5981 6000 AGTAAGGTAATCCATGAAAA 109  1998 1103843 N/A N/A 6113 6132 AGGCTCCCAAGTGAGATGTG 89 1999 1103859 N/A N/A 6214 6233 ATCACACCTTCCAGGTCAGA 68 2000 1103875 N/A N/A 6271 6290 ATATAAGCTCTGAGCTGTGG 87 2001 1103891 N/A N/A 6318 6337 TAGCCCAAATGCCCCCTCTA 101  2002 1103907 N/A N/A 6454 6473 CTCCTCAGCTAGGTGCCCTG 66 2003 1103923 N/A N/A 6534 6553 CTCCCCTGCCTGCAGGGAGG 124  2004 1103939 N/A N/A 6975 6994 ATGAACAGTGCCACAGAATC 124  2005 1103955 N/A N/A 7105 7124 ACCCCAGTTAACCCCAGGAC 90 2006 1103971 N/A N/A 7339 7358 CGTCCCCGTCCTGCCCTGGC 93 2007 1103987 N/A N/A 7473 7492 TTTCGAGGCCCGGCCCCCGG 78 2008 1104003 N/A N/A 7829 7848 AGACTCAGTCCCTGAAGGGA 89 2009 1104019 N/A N/A 7959 7978 GGCCTAATCAATATTGGTTG 80 2010 1104035 N/A N/A 8043 8062 GGTGAGGTGAGGAGTCCAAT 62 2011 1104051 N/A N/A 8116 8135 CATGTCTATCTGAAGGAAGA 159  2012 1104067 N/A N/A 8283 8302 GGACACCCCTAGGCTGGGTC 121  2013 1104083 N/A N/A 9384 9403 AAAAAGTAATTTAGCTCCCC 108  2014 1104099 N/A N/A 9419 9438 CCTTAGCTAAGAATCATTTC 76 2015 1104115 N/A N/A 9458 9477 GTCCTAAATATTCTAGTCCA 55 2016 1104131 N/A N/A 9514 9533 CAGCCTGGTTAGCCTTTCTG 38 2017 1104147 N/A N/A 9539 9558 ATTTGGCAGTATTACCTCTA 51 2018 1104163 N/A N/A 9563 9582 TTTAGTGACCTGTGACTATC 108  2019 1104179 N/A N/A 9583 9602 TCTCTGCCATTTATCTGTGC 48 2020 1104195 N/A N/A 9676 9695 ATTAACTAAAATAAAGGCTG 90 2021 1104211 N/A N/A 9811 9830 CTCAAGAAGTCCCAACTTAG 83 2022 1104227 N/A N/A 10562 10581 CCAGCACGGTATTGAAATCA 50 2023 1104243 N/A N/A 10659 10678 TAGTGCTGCTGCCAGAGTCC 54 2024 1104259 N/A N/A 10777 10796 CTCCCATGCCCGGCTTCCCC 147  2025 1104275 N/A N/A 10981 11000 TGGCCTGGCCTTGAGAATCC 108  2026 1104291 N/A N/A 11100 11119 TATGGAGGGACTGAGGAAAC 107  2027 1104307 N/A N/A 11155 11174 GTGTATTAGGATCCCATCTA 19 2028 1104323 N/A N/A 11233 11252 GTGAAAGCTACTAACTTTAA 51 2029 1104339 N/A N/A 11325 11344 ATCCCCTCTTCCCATTCCCC 128  2030 1104355 N/A N/A 11394 11413 GGCGAGTGGTAAGCTGCTGG 99 2031 1104371 N/A N/A 11525 11544 CGAGGCTGTGTAACCTTGGG 99 2032

TABLE 28 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG 14 517 1103156 142 161 3590 3609 CCCAGACGGCGGCCAGGAGC 113  2033 1103172 408 427 3856 3875 GCAGCTCAGCCTGGTAGACG 92 2034 1103188 582 601 N/A N/A CTTCATCTGCTTCCTGTCTA  23* 2035 1103204 829 848 N/A N/A AACTTGGAGCGGTACCACTC 106  2036 1103220 1249 1268 10863 10882 TCTGACACAGACTTGGTGTC 110  2037 1103236 1397 1416 11686 11705 CTGCTCGGGCCCCTCATGAG 18 2038 1103252 1534 1553 11823 11842 GTCTGGCAGGCCTGATACTG 84 2039 1103268 1682 1701 11971 11990 GGAGGGCGATGTAGTAGGTG 31 2040 1103284 1798 1817 12087 12106 CTGAGTCTCAGTTTTCCTCC 48 2041 1103300 1871 1890 12160 12179 CCTCATAAAAACCAAAAGAC 76 2042 1103316 1891 1910 12180 12199 GGCAGCATAGGGATATCCCA 74 2043 1103332 2108 2127 12397 12416 ACTGAGACACTTGAGTCATC 77 2044 1103348 2339 2358 12628 12647 CAATTGCCTCCTCCTCCATC 91 2045 1103364 2444 2463 12733 12752 GTTTCAGCATCTTCAAGAGG 63 2046 1103380 2489 2508 12778 12797 ACAAAGTCATGCCCTGCCCC 89 2047 1103396 2528 2547 12817 12836 CTGGTATGACACAGCAAGGA 67 2048 1103412 2677 2696 12966 12985 ATTTTGTGTGTGAGTAAGAA 49 2049 1103428 2752 2771 13041 13060 GAGCCAGTGTCTTCACTTTG 38 2050 1103444 3014 3033 13303 13322 GCAGTGCCCTGAAGATTAGC 88 2051 1103460 N/A N/A 8746 8765 TCCCCGTCTTTGGTGCTTTT 74 2052 1103476 N/A N/A 8823 8842 CATTTACAATCTGGTGAGCC 103  2053 1103492 N/A N/A 8981 9000 CCTGGGAAAATGACGCAGTC 88 2054 1103508 N/A N/A 9103 9122 GCTCAGAGGCCCCAGAGCAG 117  2055 1103524 N/A N/A 9242 9261 CTCTGAACTGAGTCAGCACT 93 2056 1103540 N/A N/A 9288 9307 ATAAAACTTTATTCACTGCA 82 2057 1103556 N/A N/A 8397 8416 GCCCTTCCCACGAGGCCCTG 102  2058 1103572 N/A N/A 8540 8559 GAAGTACCCTGGTATGATAG 80 2059 1103588 N/A N/A 8715 8734 TGACAAGCAGTTAAAAAAAC 98 2060 1103604 N/A N/A 4004 4023 TTCAGCCCCTTCTGCTCACA 97 2061 1103620 N/A N/A 4212 4231 TGCTTTCCCCAGTAGGGAGG 52 2062 1103636 N/A N/A 4351 4370 AATCTCCCTCATGGACATCA 102  2063 1103652 N/A N/A 4421 4440 GCAGGCAGTCACCTGTGCTT 84 2064 1103668 N/A N/A 4576 4595 AAGGCACACATGCATGTCCT 103  2065 1103684 N/A N/A 4663 4682 CTGCTTCTGCTCACACAGGC 109  2066 1103700 N/A N/A 4856 4875 CTGAGGACACTTGAATACCT 120  2067 1103716 N/A N/A 5165 5184 CTGCTTCCTGGAGTGGCAGG 123* 2068 1103732 N/A N/A 5363 5382 CTCTCCTCTCTCTGAGTGTC 100  2069 1103748 N/A N/A 5432 5451 TCTTTCCGTCTCCCTTAGTG 94 2070 1103764 N/A N/A 5518 5537 AGGGTACAGGCCACAGCTGG 101  2071 1103780 N/A N/A 5785 5804 CTTCCCCCATTCTCTTGTAC 106  2072 1103796 N/A N/A 5883 5902 AAAGCAATAGTGCCTGTGTG 80 2073 1103812 N/A N/A 5950 5969 AGAACAGAACAGTATCAGCT 96 2074 1103828 N/A N/A 5982 6001 TAGTAAGGTAATCCATGAAA 110  2075 1103844 N/A N/A 6126 6145 AGGCATGGAATGCAGGCTCC 103  2076 1103860 N/A N/A 6215 6234 TATCACACCTTCCAGGTCAG 102  2077 1103876 N/A N/A 6272 6291 CATATAAGCTCTGAGCTGTG 91 2078 1103892 N/A N/A 6319 6338 CTAGCCCAAATGCCCCCTCT 103  2079 1103908 N/A N/A 6456 6475 CACTCCTCAGCTAGGTGCCC 104  2080 1103924 N/A N/A 6535 6554 TCTCCCCTGCCTGCAGGGAG 111  2081 1103940 N/A N/A 6977 6996 GAATGAACAGTGCCACAGAA 94 2082 1103956 N/A N/A 7106 7125 CACCCCAGTTAACCCCAGGA 102  2083 1103972 N/A N/A 7340 7359 CCGTCCCCGTCCTGCCCTGG 90 2084 1103988 N/A N/A 7474 7493 GTTTCGAGGCCCGGCCCCCG 117  2085 1104004 N/A N/A 7831 7850 AAAGACTCAGTCCCTGAAGG 119  2086 1104020 N/A N/A 7960 7979 AGGCCTAATCAATATTGGTT 112  2087 1104036 N/A N/A 8044 8063 GGGTGAGGTGAGGAGTCCAA 68 2088 1104052 N/A N/A 8117 8136 GCATGTCTATCTGAAGGAAG 94 2089 1104068 N/A N/A 8289 8308 TGCCTGGGACACCCCTAGGC 122  2090 1104084 N/A N/A 9385 9404 TAAAAAGTAATTTAGCTCCC 91 2091 1104100 N/A N/A 9427 9446 ATTTTGTTCCTTAGCTAAGA 96 2092 1104116 N/A N/A 9459 9478 GGTCCTAAATATTCTAGTCC 35 2093 1104132 N/A N/A 9515 9534 TCAGCCTGGTTAGCCTTTCT 37 2094 1104148 N/A N/A 9540 9559 GATTTGGCAGTATTACCTCT 52 2095 1104164 N/A N/A 9564 9583 CTTTAGTGACCTGTGACTAT 84 2096 1104180 N/A N/A 9588 9607 ACTTCTCTCTGCCATTTATC 110  2097 1104196 N/A N/A 9677 9696 AATTAACTAAAATAAAGGCT 108  2098 1104212 N/A N/A 9820 9839 AGACCAGAGCTCAAGAAGTC 119  2099 1104228 N/A N/A 10563 10582 CCCAGCACGGTATTGAAATC 84 2100 1104244 N/A N/A 10660 10679 ATAGTGCTGCTGCCAGAGTC 88 2101 1104260 N/A N/A 10779 10798 CTCTCCCATGCCCGGCTTCC 112  2102 1104276 N/A N/A 10983 11002 CATGGCCTGGCCTTGAGAAT 31 2103 1104292 N/A N/A 11115 11134 CCCCTTAGAACAGCCTATGG 111  2104 1104308 N/A N/A 11156 11175 TGTGTATTAGGATCCCATCT 41 2105 1104324 N/A N/A 11234 11253 AGTGAAAGCTACTAACTTTA 78 2106 1104340 N/A N/A 11326 11345 AATCCCCTCTTCCCATTCCC 105  2107 1104356 N/A N/A 11396 11415 CTGGCGAGTGGTAAGCTGCT 91 2108 1104372 N/A N/A 11535 11554 TCCACCACCACGAGGCTGTG 101  2109

TABLE 29 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  8 517 1103157 158 177 3606 3625 GAGGCGGGTGCCAGGACCCA 45 2110 1103173 409 428 3857 3876 CGCAGCTCAGCCTGGTAGAC 97 2111 1103189 636 655 5225 5244 TCTCCTCCTCCAGCGACTCA 92 2112 1103205 843 862 N/A N/A CTGTCAGGTCTGCAAACTTG 74 2113 1103221 1263 1282 10877 10896 TCTTGAGGTGGCCTTCTGAC 104  2114 1103237 1399 1418 11688 11707 TTCTGCTCGGGCCCCTCATG 75 2115 1103253 1544 1563 11833 11852 GGTGGGTGCCGTCTGGCAGG 34 2116 1103269 1683 1702 11972 11991 TGGAGGGCGATGTAGTAGGT 58 2117 1103285 1800 1819 12089 12108 GTCTGAGTCTCAGTTTTCCT 25 2118 1103301 1872 1891 12161 12180 ACCTCATAAAAACCAAAAGA 116  2119 1103317 1893 1912 12182 12201 TAGGCAGCATAGGGATATCC 101  2120 1103333 2110 2129 12399 12418 GGACTGAGACACTTGAGTCA 96 2121 1103349 2348 2367 12637 12656 GCGCCATCCCAATTGCCTCC 98 2122 1103365 2446 2465 12735 12754 CTGTTTCAGCATCTTCAAGA 41 2123 1103381 2493 2512 12782 12801 TGGGACAAAGTCATGCCCTG 105  2124 1103397 2530 2549 12819 12838 GCCTGGTATGACACAGCAAG 85 2125 1103413 2697 2716 12986 13005 CTACCTAGAATACTGGGTAC 110  2126 1103429 2753 2772 13042 13061 TGAGCCAGTGTCTTCACTTT 70 2127 1103445 3015 3034 13304 13323 AGCAGTGCCCTGAAGATTAG 86 2128 1103461 N/A N/A 8748 8767 TTTCCCCGTCTTTGGTGCTT 77 2129 1103477 N/A N/A 8824 8843 CCATTTACAATCTGGTGAGC 61 2130 1103493 N/A N/A 8982 9001 TCCTGGGAAAATGACGCAGT 114  2131 1103509 N/A N/A 9142 9161 TCCCAGTGACAGGAAGAGGT 105  2132 1103525 N/A N/A 9245 9264 ATCCTCTGAACTGAGTCAGC 93 2133 1103541 N/A N/A 9292 9311 GAACATAAAACTTTATTCAC 110  2134 1103557 N/A N/A 8403 8422 TCCAGTGCCCTTCCCACGAG 104  2135 1103573 N/A N/A 8542 8561 TAGAAGTACCCTGGTATGAT 110  2136 1103589 N/A N/A 8728 8747 TTGCCCCCTGTAGTGACAAG 103  2137 1103605 N/A N/A 4005 4024 ATTCAGCCCCTTCTGCTCAC 106  2138 1103621 N/A N/A 4213 4232 CTGCTTTCCCCAGTAGGGAG 94 2139 1103637 N/A N/A 4367 4386 CTTCACCCCAGAATCCAATC 124  2140 1103653 N/A N/A 4423 4442 TGGCAGGCAGTCACCTGTGC 130  2141 1103669 N/A N/A 4577 4596 GAAGGCACACATGCATGTCC 103  2142 1103685 N/A N/A 4665 4684 ACCTGCTTCTGCTCACACAG 96 2143 1103701 N/A N/A 4858 4877 TTCTGAGGACACTTGAATAC 128  2144 1103717 N/A N/A 5166 5185 TCTGCTTCCTGGAGTGGCAG 111* 2145 1103733 N/A N/A 5364 5383 TCTCTCCTCTCTCTGAGTGT 100  2146 1103749 N/A N/A 5440 5459 CTCTTGTCTCTTTCCGTCTC 105  2147 1103765 N/A N/A 5535 5554 AAGCGGTACCAGGGCTCAGG 62 2148 1103781 N/A N/A 5810 5829 TATTCTCCCAGCTTCCTCCA 112  2149 1103797 N/A N/A 5888 5907 TTTTGAAAGCAATAGTGCCT 94 2150 1103813 N/A N/A 5951 5970 TAGAACAGAACAGTATCAGC 92 2151 1103829 N/A N/A 5989 6008 TACTTCATAGTAAGGTAATC 112  2152 1103845 N/A N/A 6144 6163 CCCAAAACAGACTGGCAGAG 79 2153 1103861 N/A N/A 6216 6235 ATATCACACCTTCCAGGTCA 87 2154 1103877 N/A N/A 6273 6292 ACATATAAGCTCTGAGCTGT 105  2155 1103893 N/A N/A 6333 6352 CGTGCCTGTCCTGCCTAGCC 90 2156 1103909 N/A N/A 6457 6476 ACACTCCTCAGCTAGGTGCC 87 2157 1103925 N/A N/A 6536 6555 TTCTCCCCTGCCTGCAGGGA 120  2158 1103941 N/A N/A 6978 6997 TGAATGAACAGTGCCACAGA 112  2159 1103957 N/A N/A 7107 7126 GCACCCCAGTTAACCCCAGG 93 2160 1103973 N/A N/A 7341 7360 CCCGTCCCCGTCCTGCCCTG 98 2161 1103989 N/A N/A 7476 7495 AGGTTTCGAGGCCCGGCCCC 93 2162 1104005 N/A N/A 7851 7870 GATCTGCACACAAGGCTGAA 99 2163 1104021 N/A N/A 7961 7980 GAGGCCTAATCAATATTGGT 111  2164 1104037 N/A N/A 8047 8066 GATGGGTGAGGTGAGGAGTC 40 2165 1104053 N/A N/A 8118 8137 CGCATGTCTATCTGAAGGAA 90 2166 1104069 N/A N/A 8325 8344 TCCTGAAAGAAAGCAGAGGG 114  2167 1104085 N/A N/A 9386 9405 GTAAAAAGTAATTTAGCTCC 127  2168 1104101 N/A N/A 9428 9447 AATTTTGTTCCTTAGCTAAG 125  2169 1104117 N/A N/A 9460 9479 TGGTCCTAAATATTCTAGTC 74 2170 1104133 N/A N/A 9516 9535 GTCAGCCTGGTTAGCCTTTC 30 2171 1104149 N/A N/A 9541 9560 GGATTTGGCAGTATTACCTC 52 2172 1104165 N/A N/A 9565 9584 GCTTTAGTGACCTGTGACTA 43 2173 1104181 N/A N/A 9589 9608 TACTTCTCTCTGCCATTTAT 75 2174 1104197 N/A N/A 9678 9697 CAATTAACTAAAATAAAGGC 130  2175 1104213 N/A N/A 9828 9847 GGATCAGGAGACCAGAGCTC 118  2176 1104229 N/A N/A 10564 10583 ACCCAGCACGGTATTGAAAT 105  2177 1104245 N/A N/A 10670 10689 CCAAATCCCAATAGTGCTGC 80 2178 1104261 N/A N/A 10782 10801 ATCCTCTCCCATGCCCGGCT 87 2179 1104277 N/A N/A 11006 11025 TGTGCCAGCCCCAGGCTTTC 125  2180 1104293 N/A N/A 11119 11138 GGCTCCCCTTAGAACAGCCT 115  2181 1104309 N/A N/A 11158 11177 AGTGTGTATTAGGATCCCAT 18 2182 1104325 N/A N/A 11235 11254 AAGTGAAAGCTACTAACTTT 102  2183 1104341 N/A N/A 11327 11346 AAATCCCCTCTTCCCATTCC 110  2184 1104357 N/A N/A 11428 11447 AGATAACACTGGGAAAGCAT 97 2185 1104373 N/A N/A 11537 11556 GGTCCACCACCACGAGGCTG 108  2186

TABLE 30 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  7 517 1103158 206 225 3654 3673 GGAGAAATCCACCCGGGTCG 155  2187 1103174 410 429 3858 3877 TCGCAGCTCAGCCTGGTAGA 127  2188 1103190 648 667 5237 5256 TCAAGAACCGGATCTCCTCC 63 2189 1103206 926 945 7283 7302 GGTCAAGGACTGCAACTGGC 96 2190 1103222 1270 1289 10884 10903 ATGTTCCTCTTGAGGTGGCC 86 2191 1103238 1400 1419 11689 11708 CTTCTGCTCGGGCCCCTCAT 59 2192 1103254 1573 1592 11862 11881 TTCTTGTTAGTTGGAGTTGC 33 2193 1103270 1686 1705 11975 11994 ATGTGGAGGGCGATGTAGTA 49 2194 1103286 1809 1828 12098 12117 CCCTTTCCTGTCTGAGTCTC 72 2195 1103302 1873 1892 12162 12181 CACCTCATAAAAACCAAAAG 100  2196 1103318 1996 2015 12285 12304 GAACAACCCTCTGAGCTGGG 57 2197 1103334 2124 2143 12413 12432 ATGGCAGCTCAGGTGGACTG 54 2198 1103350 2364 2383 12653 12672 CCTACTTGTATGCCTAGCGC 75 2199 1103366 2447 2466 12736 12755 CCTGTTTCAGCATCTTCAAG 42 2200 1103382 2494 2513 12783 12802 ATGGGACAAAGTCATGCCCT 98 2201 1103398 2567 2586 12856 12875 AAGAAGCAGCAGTCCCAGGG 75 2202 1103414 2700 2719 12989 13008 GCACTACCTAGAATACTGGG 76 2203 1103430 2754 2773 13043 13062 ATGAGCCAGTGTCTTCACTT 37 2204 1103446 3017 3036 13306 13325 GCAGCAGTGCCCTGAAGATT 29 2205 1103462 N/A N/A 8749 8768 TTTTCCCCGTCTTTGGTGCT 56 2206 1103478 N/A N/A 8825 8844 TCCATTTACAATCTGGTGAG 41 2207 1103494 N/A N/A 8983 9002 TTCCTGGGAAAATGACGCAG 97 2208 1103510 N/A N/A 9144 9163 CCTCCCAGTGACAGGAAGAG 104  2209 1103526 N/A N/A 9246 9265 AATCCTCTGAACTGAGTCAG 54 2210 1103542 N/A N/A 9293 9312 GGAACATAAAACTTTATTCA 69 2211 1103558 N/A N/A 8405 8424 ACTCCAGTGCCCTTCCCACG 27 2212 1103574 N/A N/A 8543 8562 CTAGAAGTACCCTGGTATGA 95 2213 1103590 N/A N/A 8730 8749 TTTTGCCCCCTGTAGTGACA 67 2214 1103606 N/A N/A 4038 4057 TCCCTGAGACTTCTCGGGCA 110  2215 1103622 N/A N/A 4214 4233 ACTGCTTTCCCCAGTAGGGA 77 2216 1103638 N/A N/A 4371 4390 CTTTCTTCACCCCAGAATCC 139  2217 1103654 N/A N/A 4424 4443 GTGGCAGGCAGTCACCTGTG 83 2218 1103670 N/A N/A 4578 4597 TGAAGGCACACATGCATGTC 105  2219 1103686 N/A N/A 4666 4685 CACCTGCTTCTGCTCACACA 158  2220 1103702 N/A N/A 4960 4979 CCTGACCTGTCTATAGGCAG  92* 2221 1103718 N/A N/A 5167 5186 ATCTGCTTCCTGGAGTGGCA 102* 2222 1103734 N/A N/A 5365 5384 TTCTCTCCTCTCTCTGAGTG 102  2223 1103750 N/A N/A 5442 5461 CTCTCTTGTCTCTTTCCGTC 69 2224 1103766 N/A N/A 5536 5555 GAAGCGGTACCAGGGCTCAG 95 2225 1103782 N/A N/A 5811 5830 ATATTCTCCCAGCTTCCTCC 92 2226 1103798 N/A N/A 5893 5912 GGTACTTTTGAAAGCAATAG 47 2227 1103814 N/A N/A 5955 5974 GGCTTAGAACAGAACAGTAT 87 2228 1103830 N/A N/A 5996 6015 CAGCACCTACTTCATAGTAA 54 2229 1103846 N/A N/A 6156 6175 GAGGCTCAGTAACCCAAAAC 60 2230 1103862 N/A N/A 6217 6236 AATATCACACCTTCCAGGTC 130  2231 1103878 N/A N/A 6275 6294 CTACATATAAGCTCTGAGCT 127  2232 1103894 N/A N/A 6369 6388 AGGAATGGCCCTCCCTTCTT 93 2233 1103910 N/A N/A 6458 6477 CACACTCCTCAGCTAGGTGC 75 2234 1103926 N/A N/A 6541 6560 GCCCTTTCTCCCCTGCCTGC 106  2235 1103942 N/A N/A 6980 6999 AATGAATGAACAGTGCCACA 113  2236 1103958 N/A N/A 7114 7133 GTGAGCAGCACCCCAGTTAA 117  2237 1103974 N/A N/A 7345 7364 TCCGCCCGTCCCCGTCCTGC 119  2238 1103990 N/A N/A 7477 7496 GAGGTTTCGAGGCCCGGCCC 129  2239 1104006 N/A N/A 7852 7871 GGATCTGCACACAAGGCTGA 112  2240 1104022 N/A N/A 7962 7981 TGAGGCCTAATCAATATTGG 96 2241 1104038 N/A N/A 8050 8069 AGAGATGGGTGAGGTGAGGA 36 2242 1104054 N/A N/A 8140 8159 CTTGAGTGTTATCTGGGAGG 61 2243 1104070 N/A N/A 8336 8355 GGGAATGGTGATCCTGAAAG 53 2244 1104086 N/A N/A 9388 9407 AAGTAAAAAGTAATTTAGCT 87 2245 1104102 N/A N/A 9429 9448 GAATTTTGTTCCTTAGCTAA 80 2246 1104118 N/A N/A 9477 9496 GAGACATGCATATCTAGTGG 23 2247 1104134 N/A N/A 9518 9537 TAGTCAGCCTGGTTAGCCTT 63 2248 1104150 N/A N/A 9547 9566 TATCTAGGATTTGGCAGTAT 58 2249 1104166 N/A N/A 9567 9586 GTGCTTTAGTGACCTGTGAC 25 2250 1104182 N/A N/A 9591 9610 CCTACTTCTCTCTGCCATTT 75 2251 1104198 N/A N/A 9679 9698 ACAATTAACTAAAATAAAGG 95 2252 1104214 N/A N/A 10003 10022 GTAATCCCCTTACTCGGGAG 84 2253 1104230 N/A N/A 10565 10584 AACCCAGCACGGTATTGAAA 117  2254 1104246 N/A N/A 10671 10690 CCCAAATCCCAATAGTGCTG 59 2255 1104262 N/A N/A 10786 10805 AGCCATCCTCTCCCATGCCC 130  2256 1104278 N/A N/A 11010 11029 TCTATGTGCCAGCCCCAGGC 83 2257 1104294 N/A N/A 11120 11139 AGGCTCCCCTTAGAACAGCC 75 2258 1104310 N/A N/A 11159 11178 GAGTGTGTATTAGGATCCCA 18 2259 1104326 N/A N/A 11237 11256 GGAAGTGAAAGCTACTAACT 46 2260 1104342 N/A N/A 11329 11348 CCAAATCCCCTCTTCCCATT 81 2261 1104358 N/A N/A 11430 11449 TGAGATAACACTGGGAAAGC 89 2262 1104374 N/A N/A 11543 11562 ACCCCAGGTCCACCACCACG 101  2263

TABLE 31 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  7 517 1103159 218 237 3666 3685 TGCCCCAGCCAGGGAGAAAT 93 2264 1103175 411 430 3859 3878 CTCGCAGCTCAGCCTGGTAG 69 2265 1103191 652 671 5241 5260 TTCCTCAAGAACCGGATCTC 79 2266 1103207 930 949 7287 7306 CGCAGGTCAAGGACTGCAAC 84 2267 1103223 1271 1290 10885 10904 GATGTTCCTCTTGAGGTGGC 94 2268 1103239 1401 1420 11690 11709 GCTTCTGCTCGGGCCCCTCA 52 2269 1103255 1575 1594 11864 11883 GTTTCTTGTTAGTTGGAGTT 19 2270 1103271 1687 1706 11976 11995 GATGTGGAGGGCGATGTAGT 69 2271 1103287 1811 1830 12100 12119 TTCCCTTTCCTGTCTGAGTC 56 2272 1103303 1874 1893 12163 12182 CCACCTCATAAAAACCAAAA 89 2273 1103319 1997 2016 12286 12305 AGAACAACCCTCTGAGCTGG 61 2274 1103335 2183 2202 12472 12491 CAAGTGCTGAGAATCAAGCT 87 2275 1103351 2398 2417 12687 12706 TCCAGAGGCCAAGTGCAACT 57 2276 1103367 2448 2467 12737 12756 TCCTGTTTCAGCATCTTCAA 61 2277 1103383 2495 2514 12784 12803 AATGGGACAAAGTCATGCCC 88 2278 1103399 2629 2648 12918 12937 AAAAGCAGCCGGTCACTATG 100  2279 1103415 2703 2722 12992 13011 AGGGCACTACCTAGAATACT 71 2280 1103431 2789 2808 13078 13097 CAGCCCTGAGCACCCGGCCT 106  2281 1103447 3019 3038 13308 13327 CAGCAGCAGTGCCCTGAAGA 38 2282 1103463 N/A N/A 8750 8769 ATTTTCCCCGTCTTTGGTGC 65 2283 1103479 N/A N/A 8855 8874 GGCAGGCAGCTAACCGCGAG 80 2284 1103495 N/A N/A 8984 9003 GTTCCTGGGAAAATGACGCA 89 2285 1103511 N/A N/A 9146 9165 GCCCTCCCAGTGACAGGAAG 96 2286 1103527 N/A N/A 9249 9268 AAGAATCCTCTGAACTGAGT 85 2287 1103543 N/A N/A 9294 9313 GGGAACATAAAACTTTATTC 42 2288 1103559 N/A N/A 8407 8426 GGACTCCAGTGCCCTTCCCA 90 2289 1103575 N/A N/A 8544 8563 CCTAGAAGTACCCTGGTATG 64 2290 1103591 N/A N/A 8732 8751 GCTTTTGCCCCCTGTAGTGA 36 2291 1103607 N/A N/A 4039 4058 CTCCCTGAGACTTCTCGGGC 129  2292 1103623 N/A N/A 4216 4235 GCACTGCTTTCCCCAGTAGG 88 2293 1103639 N/A N/A 4372 4391 ACTTTCTTCACCCCAGAATC 101  2294 1103655 N/A N/A 4456 4475 AGTCAAAGTAACTTGATGGG 90 2295 1103671 N/A N/A 4579 4598 TTGAAGGCACACATGCATGT 102  2296 1103687 N/A N/A 4728 4747 AGGATGAGCAGATGTGGGCT 77 2297 1103703 N/A N/A 4961 4980 CCCTGACCTGTCTATAGGCA 105* 2298 1103719 N/A N/A 5169 5188 TCATCTGCTTCCTGGAGTGG  49* 2299 1103735 N/A N/A 5366 5385 TTTCTCTCCTCTCTCTGAGT 95 2300 1103751 N/A N/A 5446 5465 AATGCTCTCTTGTCTCTTTC 83 2301 1103767 N/A N/A 5537 5556 AGAAGCGGTACCAGGGCTCA 100  2302 1103783 N/A N/A 5830 5849 AGTCAGTCACCTGGAGAGGA 79 2303 1103799 N/A N/A 5898 5917 TAATGGGTACTTTTGAAAGC 100  2304 1103815 N/A N/A 5956 5975 GGGCTTAGAACAGAACAGTA 74 2305 1103831 N/A N/A 5997 6016 ACAGCACCTACTTCATAGTA 113  2306 1103847 N/A N/A 6158 6177 TAGAGGCTCAGTAACCCAAA 74 2307 1103863 N/A N/A 6218 6237 CAATATCACACCTTCCAGGT 72 2308 1103879 N/A N/A 6277 6296 CACTACATATAAGCTCTGAG 93 2309 1103895 N/A N/A 6370 6389 TAGGAATGGCCCTCCCTTCT 91 2310 1103911 N/A N/A 6461 6480 ACTCACACTCCTCAGCTAGG 100  2311 1103927 N/A N/A 6546 6565 TAATAGCCCTTTCTCCCCTG 93 2312 1103943 N/A N/A 6982 7001 CGAATGAATGAACAGTGCCA 108  2313 1103959 N/A N/A 7120 7139 AGGGAGGTGAGCAGCACCCC 103  2314 1103975 N/A N/A 7347 7366 GCTCCGCCCGTCCCCGTCCT 66 2315 1103991 N/A N/A 7478 7497 GGAGGTTTCGAGGCCCGGCC 117  2316 1104007 N/A N/A 7853 7872 GGGATCTGCACACAAGGCTG 49 2317 1104023 N/A N/A 7963 7982 TTGAGGCCTAATCAATATTG 110  2318 1104039 N/A N/A 8052 8071 GAAGAGATGGGTGAGGTGAG 36 2319 1104055 N/A N/A 8193 8212 CTTTTTCCCCAGCAGCCAAC 125  2320 1104071 N/A N/A 9325 9344 TCACATTCACTAATATTTAA 74 2321 1104087 N/A N/A 9389 9408 CAAGTAAAAAGTAATTTAGC 120  2322 1104103 N/A N/A 9433 9452 AGAGGAATTTTGTTCCTTAG 98 2323 1104119 N/A N/A 9481 9500 TCCTGAGACATGCATATCTA 82 2324 1104135 N/A N/A 9520 9539 ACTAGTCAGCCTGGTTAGCC 66 2325 1104151 N/A N/A 9549 9568 ACTATCTAGGATTTGGCAGT 36 2326 1104167 N/A N/A 9569 9588 CTGTGCTTTAGTGACCTGTG 42 2327 1104183 N/A N/A 9636 9655 AGTGCTCAATACACATAGGT 58 2328 1104199 N/A N/A 9680 9699 GACAATTAACTAAAATAAAG 87 2329 1104215 N/A N/A 10131 10150 ACTCCGTCGAAAGCAGGCAA 91 2330 1104231 N/A N/A 10566 10585 AAACCCAGCACGGTATTGAA 71 2331 1104247 N/A N/A 10694 10713 TCTTCCAAACGGGCTGGAGA 98 2332 1104263 N/A N/A 10790 10809 CATGAGCCATCCTCTCCCAT 70 2333 1104279 N/A N/A 11023 11042 TTGCTGGGAACCTTCTATGT 97 2334 1104295 N/A N/A 11121 11140 AAGGCTCCCCTTAGAACAGC 64 2335 1104311 N/A N/A 11160 11179 AGAGTGTGTATTAGGATCCC 31 2336 1104327 N/A N/A 11242 11261 GAGTTGGAAGTGAAAGCTAC 67 2337 1104343 N/A N/A 11342 11361 CGTGGCGGATACGCCAAATC 96 2338 1104359 N/A N/A 11431 11450 GTGAGATAACACTGGGAAAG 65 2339 1104375 N/A N/A 11552 11571 TTCACACAGACCCCAGGTCC 96 2340

TABLE 32 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  6 517 1103160 221 240 3669 3688 GAGTGCCCCAGCCAGGGAGA 119  2341 1103176 442 461 3890 3909 GCGGTGAGTTGATCGAGCCG 86 2342 1103192 653 672 5242 5261 CTTCCTCAAGAACCGGATCT 81 2343 1103208 932 951 7289 7308 GTCGCAGGTCAAGGACTGCA 100  2344 1103224 1293 1312 10907 10926 GCATCTCCACGGTCTTCACC 85 2345 1103240 1403 1422 11692 11711 CTGCTTCTGCTCGGGCCCCT 80 2346 1103256 1577 1596 11866 11885 GAGTTTCTTGTTAGTTGGAG  9 2347 1103272 1700 1719 11989 12008 AACAGGAATCAGGGATGTGG 35 2348 1103288 1836 1855 12125 12144 CCAGGGCTACCTTGTCTGTG 90 2349 1103304 1878 1897 12167 12186 TATCCCACCTCATAAAAACC 88 2350 1103320 2001 2020 12290 12309 TAGGAGAACAACCCTCTGAG 62 2351 1103336 2185 2204 12474 12493 CCCAAGTGCTGAGAATCAAG 53 2352 1103352 2400 2419 12689 12708 AATCCAGAGGCCAAGTGCAA 95 2353 1103368 2452 2471 12741 12760 TCTCTCCTGTTTCAGCATCT 22 2354 1103384 2496 2515 12785 12804 AAATGGGACAAAGTCATGCC 98 2355 1103400 2637 2656 12926 12945 GCTTAGGGAAAAGCAGCCGG 51 2356 1103416 2704 2723 12993 13012 TAGGGCACTACCTAGAATAC 75 2357 1103432 2791 2810 13080 13099 GTCAGCCCTGAGCACCCGGC 116  2358 1103448 3021 3040 13310 13329 GGCAGCAGCAGTGCCCTGAA 38 2359 1103464 N/A N/A 8752 8771 TGATTTTCCCCGTCTTTGGT 58 2360 1103480 N/A N/A 8866 8885 CGTGTCTGAGAGGCAGGCAG 76 2361 1103496 N/A N/A 8989 9008 CTGCAGTTCCTGGGAAAATG 70 2362 1103512 N/A N/A 9147 9166 GGCCCTCCCAGTGACAGGAA 68 2363 1103528 N/A N/A 9250 9269 GAAGAATCCTCTGAACTGAG 64 2364 1103544 N/A N/A 9295 9314 AGGGAACATAAAACTTTATT 57 2365 1103560 N/A N/A 8408 8427 AGGACTCCAGTGCCCTTCCC 57 2366 1103576 N/A N/A 8546 8565 CACCTAGAAGTACCCTGGTA 101  2367 1103592 N/A N/A 8734 8753 GTGCTTTTGCCCCCTGTAGT 43 2368 1103608 N/A N/A 4040 4059 CCTCCCTGAGACTTCTCGGG 86 2369 1103624 N/A N/A 4217 4236 TGCACTGCTTTCCCCAGTAG 102  2370 1103640 N/A N/A 4373 4392 CACTTTCTTCACCCCAGAAT 135  2371 1103656 N/A N/A 4465 4484 AGCTGTGCAAGTCAAAGTAA 80 2372 1103672 N/A N/A 4584 4603 TGCACTTGAAGGCACACATG 59 2373 1103688 N/A N/A 4729 4748 GAGGATGAGCAGATGTGGGC 65 2374 1103704 N/A N/A 4962 4981 TCCCTGACCTGTCTATAGGC  95* 2375 1103720 N/A N/A 5170 5189 TTCATCTGCTTCCTGGAGTG  72* 2376 1103736 N/A N/A 5381 5400 ACCTGCCAATCTCTGTTTCT 94 2377 1103752 N/A N/A 5447 5466 GAATGCTCTCTTGTCTCTTT 54 2378 1103768 N/A N/A 5541 5560 TGAGAGAAGCGGTACCAGGG 79 2379 1103784 N/A N/A 5832 5851 AAAGTCAGTCACCTGGAGAG 87 2380 1103800 N/A N/A 5907 5926 CAGTAATAATAATGGGTACT 67 2381 1103816 N/A N/A 5957 5976 AGGGCTTAGAACAGAACAGT 50 2382 1103832 N/A N/A 6005 6024 GTCAAAGAACAGCACCTACT 121  2383 1103848 N/A N/A 6160 6179 GGTAGAGGCTCAGTAACCCA 68 2384 1103864 N/A N/A 6219 6238 TCAATATCACACCTTCCAGG 80 2385 1103880 N/A N/A 6278 6297 ACACTACATATAAGCTCTGA 109  2386 1103896 N/A N/A 6382 6401 TCTGTCCTCCACTAGGAATG 120  2387 1103912 N/A N/A 6462 6481 CACTCACACTCCTCAGCTAG 85 2388 1103928 N/A N/A 6554 6573 CTGGGTTCTAATAGCCCTTT 77 2389 1103944 N/A N/A 6983 7002 GCGAATGAATGAACAGTGCC 75 2390 1103960 N/A N/A 7122 7141 TCAGGGAGGTGAGCAGCACC 111  2391 1103976 N/A N/A 7354 7373 CGTCCCTGCTCCGCCCGTCC 93 2392 1103992 N/A N/A 7504 7523 TCCCGCGGAGCCCCGACCCG 90 2393 1104008 N/A N/A 7857 7876 GGAAGGGATCTGCACACAAG 59 2394 1104024 N/A N/A 7965 7984 CCTTGAGGCCTAATCAATAT 80 2395 1104040 N/A N/A 8053 8072 AGAAGAGATGGGTGAGGTGA 85 2396 1104056 N/A N/A 8201 8220 TCTCCTAGCTTTTTCCCCAG 131  2397 1104072 N/A N/A 9327 9346 CGTCACATTCACTAATATTT 22 2398 1104088 N/A N/A 9395 9414 CCAATGCAAGTAAAAAGTAA 92 2399 1104104 N/A N/A 9434 9453 AAGAGGAATTTTGTTCCTTA 72 2400 1104120 N/A N/A 9483 9502 AGTCCTGAGACATGCATATC 77 2401 1104136 N/A N/A 9521 9540 TACTAGTCAGCCTGGTTAGC 69 2402 1104152 N/A N/A 9550 9569 GACTATCTAGGATTTGGCAG 22 2403 1104168 N/A N/A 9570 9589 TCTGTGCTTTAGTGACCTGT 56 2404 1104184 N/A N/A 9637 9656 TAGTGCTCAATACACATAGG 58 2405 1104200 N/A N/A 9681 9700 AGACAATTAACTAAAATAAA 85 2406 1104216 N/A N/A 10132 10151 GACTCCGTCGAAAGCAGGCA 63 2407 1104232 N/A N/A 10570 10589 CTGAAAACCCAGCACGGTAT 142  2408 1104248 N/A N/A 10695 10714 CTCTTCCAAACGGGCTGGAG 69 2409 1104264 N/A N/A 10791 10810 GCATGAGCCATCCTCTCCCA 121  2410 1104280 N/A N/A 11024 11043 GTTGCTGGGAACCTTCTATG 34 2411 1104296 N/A N/A 11122 11141 CAAGGCTCCCCTTAGAACAG 56 2412 1104312 N/A N/A 11162 11181 AGAGAGTGTGTATTAGGATC 37 2413 1104328 N/A N/A 11243 11262 AGAGTTGGAAGTGAAAGCTA 73 2414 1104344 N/A N/A 11370 11389 AGATGAGCTCCCACTGTGGT 74 2415 1104360 N/A N/A 11432 11451 GGTGAGATAACACTGGGAAA 48 2416 1104376 N/A N/A 11553 11572 GTTCACACAGACCCCAGGTC 96 2417

TABLE 33 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  5 517 1103161 222 241 3670 3689 TGAGTGCCCCAGCCAGGGAG 168  2418 1103177 443 462 3891 3910 GGCGGTGAGTTGATCGAGCC 101  2419 1103193 654 673 5243 5262 TCTTCCTCAAGAACCGGATC 68 2420 1103209 948 967 7305 7324 CGCGCAGAGACTCCAGGTCG 112  2421 1103225 1295 1314 10909 10928 CCGCATCTCCACGGTCTTCA 74 2422 1103241 1409 1428 11698 11717 ACTATCCTGCTTCTGCTCGG 41 2423 1103257 1578 1597 11867 11886 TGAGTTTCTTGTTAGTTGGA 24 2424 1103273 1702 1721 11991 12010 ACAACAGGAATCAGGGATGT 94 2425 1103289 1840 1859 12129 12148 CTGGCCAGGGCTACCTTGTC 95 2426 1103305 1879 1898 12168 12187 ATATCCCACCTCATAAAAAC 108  2427 1103321 2013 2032 12302 12321 TCAGGGTCAGTCTAGGAGAA 63 2428 1103337 2189 2208 12478 12497 ATCCCCCAAGTGCTGAGAAT 96 2429 1103353 2403 2422 12692 12711 CACAATCCAGAGGCCAAGTG 70 2430 1103369 2453 2472 12742 12761 TTCTCTCCTGTTTCAGCATC 32 2431 1103385 2497 2516 12786 12805 GAAATGGGACAAAGTCATGC 79 2432 1103401 2638 2657 12927 12946 GGCTTAGGGAAAAGCAGCCG 140  2433 1103417 2705 2724 12994 13013 ATAGGGCACTACCTAGAATA 117  2434 1103433 2792 2811 13081 13100 TGTCAGCCCTGAGCACCCGG 64 2435 1103449 3031 3050 13320 13339 AGCGACTAAAGGCAGCAGCA 64 2436 1103465 N/A N/A 8753 8772 GTGATTTTCCCCGTCTTTGG 26 2437 1103481 N/A N/A 8892 8911 ACTCCCTGTCAAGCTGGGCA 110  2438 1103497 N/A N/A 8991 9010 CACTGCAGTTCCTGGGAAAA 92 2439 1103513 N/A N/A 9170 9189 TCCAGGCACAGCGAGACCCA 121  2440 1103529 N/A N/A 9251 9270 GGAAGAATCCTCTGAACTGA 86 2441 1103545 N/A N/A 9296 9315 CAGGGAACATAAAACTTTAT 135  2442 1103561 N/A N/A 8409 8428 CAGGACTCCAGTGCCCTTCC 97 2443 1103577 N/A N/A 8548 8567 CCCACCTAGAAGTACCCTGG 72 2444 1103593 N/A N/A 8736 8755 TGGTGCTTTTGCCCCCTGTA 84 2445 1103609 N/A N/A 4044 4063 GGCCCCTCCCTGAGACTTCT 161  2446 1103625 N/A N/A 4218 4237 CTGCACTGCTTTCCCCAGTA 123  2447 1103641 N/A N/A 4381 4400 CTTTCCCTCACTTTCTTCAC 135  2448 1103657 N/A N/A 4479 4498 GACCACCGCTTCACAGCTGT 118  2449 1103673 N/A N/A 4594 4613 CATGTCCTCCTGCACTTGAA 66 2450 1103689 N/A N/A 4745 4764 ACAGAGGACTTGTCTGGAGG 77 2451 1103705 N/A N/A 4963 4982 CTCCCTGACCTGTCTATAGG  97* 2452 1103721 N/A N/A 5171 5190 CTTCATCTGCTTCCTGGAGT  78* 2453 1103737 N/A N/A 5382 5401 TACCTGCCAATCTCTGTTTC 79 2454 1103753 N/A N/A 5449 5468 TCGAATGCTCTCTTGTCTCT 92 2455 1103769 N/A N/A 5543 5562 GGTGAGAGAAGCGGTACCAG 141  2456 1103785 N/A N/A 5834 5853 TGAAAGTCAGTCACCTGGAG 102  2457 1103801 N/A N/A 5908 5927 GCAGTAATAATAATGGGTAC 69 2458 1103817 N/A N/A 5958 5977 CAGGGCTTAGAACAGAACAG 61 2459 1103833 N/A N/A 6014 6033 CTCTCATCTGTCAAAGAACA 87 2460 1103849 N/A N/A 6161 6180 TGGTAGAGGCTCAGTAACCC 60 2461 1103865 N/A N/A 6246 6265 TCTACGGGCACTATGTTTGG 79 2462 1103881 N/A N/A 6279 6298 CACACTACATATAAGCTCTG 188  2463 1103897 N/A N/A 6398 6417 TTTTCTGCCTCCAGGCTCTG 93 2464 1103913 N/A N/A 6469 6488 CTTCTGCCACTCACACTCCT 101  2465 1103929 N/A N/A 6806 6825 GTCAGTGGCACAATCCCGGG 76 2466 1103945 N/A N/A 6987 7006 GCAAGCGAATGAATGAACAG 76 2467 1103961 N/A N/A 7123 7142 ATCAGGGAGGTGAGCAGCAC 51 2468 1103977 N/A N/A 7360 7379 GGTGGCCGTCCCTGCTCCGC 89 2469 1103993 N/A N/A 7760 7779 TGATGAGGGCTCACCGGTTC 77 2470 1104009 N/A N/A 7896 7915 GCTATGTGTGAGGCAGGCAC 138  2471 1104025 N/A N/A 7979 7998 ACCCAAGTCCTTGGCCTTGA 79 2472 1104041 N/A N/A 8054 8073 CAGAAGAGATGGGTGAGGTG 71 2473 1104057 N/A N/A 8202 8221 ATCTCCTAGCTTTTTCCCCA 147  2474 1104073 N/A N/A 9345 9364 AGAGAGAAAAATATAACACG 90 2475 1104089 N/A N/A 9401 9420 TCAGGGCCAATGCAAGTAAA 53 2476 1104105 N/A N/A 9436 9455 AGAAGAGGAATTTTGTTCCT 68 2477 1104121 N/A N/A 9484 9503 AAGTCCTGAGACATGCATAT 64 2478 1104137 N/A N/A 9522 9541 CTACTAGTCAGCCTGGTTAG 68 2479 1104153 N/A N/A 9551 9570 TGACTATCTAGGATTTGGCA 38 2480 1104169 N/A N/A 9571 9590 ATCTGTGCTTTAGTGACCTG 80 2481 1104185 N/A N/A 9638 9657 TTAGTGCTCAATACACATAG 75 2482 1104201 N/A N/A 9682 9701 GAGACAATTAACTAAAATAA 86 2483 1104217 N/A N/A 10133 10152 AGACTCCGTCGAAAGCAGGC 78 2484 1104233 N/A N/A 10571 10590 TCTGAAAACCCAGCACGGTA 116  2485 1104249 N/A N/A 10696 10715 GCTCTTCCAAACGGGCTGGA 116  2486 1104265 N/A N/A 10795 10814 CAGGGCATGAGCCATCCTCT 101  2487 1104281 N/A N/A 11056 11075 CTCCTCCAGAATTCCCTGGG 140  2488 1104297 N/A N/A 11130 11149 TTTGGTACCAAGGCTCCCCT 72 2489 1104313 N/A N/A 11178 11197 TTTGAGGGTGAGAAAGAGAG 82 2490 1104329 N/A N/A 11259 11278 CCCAACTGTGTCTGCTAGAG 47 2491 1104345 N/A N/A 11371 11390 AAGATGAGCTCCCACTGTGG 111  2492 1104361 N/A N/A 11434 11453 GTGGTGAGATAACACTGGGA 43 2493 1104377 N/A N/A 11557 11576 AGGAGTTCACACAGACCCCA 92 2494

TABLE 34 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  6 517 1103162 224 243 3672 3691 ATTGAGTGCCCCAGCCAGGG 94 2495 1103178 468 487 3916 3935 TCTCAACCTCCAGCCGGGCG  23* 2496 1103194 687 706 5573 5592 GCTCCTGGAGTTCCCGAACC 139  2497 1103210 949 968 7306 7325 CCGCGCAGAGACTCCAGGTC 98 2498 1103226 1316 1335 N/A N/A CTCCTTAATGACCTCTCCAT 95 2499 1103242 1419 1438 11708 11727 AGGCGGAGCAACTATCCTGC 150  2500 1103258 1579 1598 11868 11887 GTGAGTTTCTTGTTAGTTGG 35 2501 1103274 1706 1725 11995 12014 CATAACAACAGGAATCAGGG 49 2502 1103290 1846 1865 12135 12154 AAGCCTCTGGCCAGGGCTAC 113  2503 1103306 1880 1899 12169 12188 GATATCCCACCTCATAAAAA 98 2504 1103322 2015 2034 12304 12323 GATCAGGGTCAGTCTAGGAG 46 2505 1103338 2196 2215 12485 12504 ACAACAGATCCCCCAAGTGC 89 2506 1103354 2405 2424 12694 12713 CCCACAATCCAGAGGCCAAG 79 2507 1103370 2454 2473 12743 12762 TTTCTCTCCTGTTTCAGCAT 31 2508 1103386 2498 2517 12787 12806 AGAAATGGGACAAAGTCATG 102  2509 1103402 2643 2662 12932 12951 CCCTTGGCTTAGGGAAAAGC 102  2510 1103418 2706 2725 12995 13014 AATAGGGCACTACCTAGAAT 130  2511 1103434 2793 2812 13082 13101 GTGTCAGCCCTGAGCACCCG 73 2512 1103450 3032 3051 13321 13340 CAGCGACTAAAGGCAGCAGC 76 2513 1103466 N/A N/A 8759 8778 GACCTTGTGATTTTCCCCGT 55 2514 1103482 N/A N/A 8893 8912 CACTCCCTGTCAAGCTGGGC 112  2515 1103498 N/A N/A 9028 9047 TAATGTACAGTTACTCTGTA 80 2516 1103514 N/A N/A 9206 9225 CCTCAGGGATGAAAGAATAA 56 2517 1103530 N/A N/A 9252 9271 GGGAAGAATCCTCTGAACTG 83 2518 1103546 N/A N/A 9297 9316 GCAGGGAACATAAAACTTTA 132  2519 1103562 N/A N/A 8441 8460 CTGGAGCAACCTACAGGCCC 103  2520 1103578 N/A N/A 8550 8569 GCCCCACCTAGAAGTACCCT 83 2521 1103594 N/A N/A 8738 8757 TTTGGTGCTTTTGCCCCCTG 76 2522 1103610 N/A N/A 4045 4064 CGGCCCCTCCCTGAGACTTC 92 2523 1103626 N/A N/A 4219 4238 CCTGCACTGCTTTCCCCAGT 124  2524 1103642 N/A N/A 4384 4403 GCTCTTTCCCTCACTTTCTT 111  2525 1103658 N/A N/A 4480 4499 TGACCACCGCTTCACAGCTG 66 2526 1103674 N/A N/A 4598 4617 CGCACATGTCCTCCTGCACT 122  2527 1103690 N/A N/A 4746 4765 GACAGAGGACTTGTCTGGAG 69 2528 1103706 N/A N/A 4966 4985 CACCTCCCTGACCTGTCTAT 165* 2529 1103722 N/A N/A 5173 5192 GGCTTCATCTGCTTCCTGGA  11* 2530 1103738 N/A N/A 5390 5409 CCTGTCTCTACCTGCCAATC 93 2531 1103754 N/A N/A 5456 5475 CAGGAGTTCGAATGCTCTCT 77 2532 1103770 N/A N/A 5555 5574 CCTCCTGACCAGGGTGAGAG 91 2533 1103786 N/A N/A 5838 5857 CTGGTGAAAGTCAGTCACCT 84 2534 1103802 N/A N/A 5909 5928 AGCAGTAATAATAATGGGTA 122  2535 1103818 N/A N/A 5960 5979 CACAGGGCTTAGAACAGAAC 101  2536 1103834 N/A N/A 6015 6034 TCTCTCATCTGTCAAAGAAC 81 2537 1103850 N/A N/A 6162 6181 ATGGTAGAGGCTCAGTAACC 66 2538 1103866 N/A N/A 6247 6266 CTCTACGGGCACTATGTTTG 72 2539 1103882 N/A N/A 6286 6305 TGGCTCCCACACTACATATA 89 2540 1103898 N/A N/A 6401 6420 TGCTTTTCTGCCTCCAGGCT 82 2541 1103914 N/A N/A 6476 6495 CCAGTGGCTTCTGCCACTCA 92 2542 1103930 N/A N/A 6898 6917 AGCGGAGGCCTGGGTGTTTT 74 2543 1103946 N/A N/A 6988 7007 AGCAAGCGAATGAATGAACA 116  2544 1103962 N/A N/A 7124 7143 AATCAGGGAGGTGAGCAGCA 71 2545 1103978 N/A N/A 7384 7403 CCCTTCTCCCCTGGCATCTC 130  2546 1103994 N/A N/A 7792 7811 GTGAGGCAGCAGGGAGACTT 80 2547 1104010 N/A N/A 7904 7923 GACTGCCTGCTATGTGTGAG 118  2548 1104026 N/A N/A 7981 8000 TGACCCAAGTCCTTGGCCTT 109  2549 1104042 N/A N/A 8056 8075 GGCAGAAGAGATGGGTGAGG 75 2550 1104058 N/A N/A 8207 8226 ACTCCATCTCCTAGCTTTTT 95 2551 1104074 N/A N/A 9347 9366 CGAGAGAGAAAAATATAACA 129  2552 1104090 N/A N/A 9403 9422 TTTCAGGGCCAATGCAAGTA 49 2553 1104106 N/A N/A 9447 9466 TCTAGTCCAGAAGAAGAGGA 76 2554 1104122 N/A N/A 9494 9513 ATGCTGAATTAAGTCCTGAG 38 2555 1104138 N/A N/A 9523 9542 TCTACTAGTCAGCCTGGTTA 68 2556 1104154 N/A N/A 9553 9572 TGTGACTATCTAGGATTTGG 68 2557 1104170 N/A N/A 9572 9591 TATCTGTGCTTTAGTGACCT 53 2558 1104186 N/A N/A 9639 9658 ATTAGTGCTCAATACACATA 101  2559 1104202 N/A N/A 9683 9702 GGAGACAATTAACTAAAATA 94 2560 1104218 N/A N/A 10135 10154 TAAGACTCCGTCGAAAGCAG 131  2561 1104234 N/A N/A 10572 10591 TTCTGAAAACCCAGCACGGT 110  2562 1104250 N/A N/A 10708 10727 GAGAGAGCCTAGGCTCTTCC 70 2563 1104266 N/A N/A 10796 10815 TCAGGGCATGAGCCATCCTC 60 2564 1104282 N/A N/A 11065 11084 TTCCTTGCTCTCCTCCAGAA 156  2565 1104298 N/A N/A 11144 11163 TCCCATCTAGTGGCTTTGGT 48 2566 1104314 N/A N/A 11212 11231 TCTCTTTCTCTCCCTGGCAA 74 2567 1104330 N/A N/A 11264 11283 CCATCCCCAACTGTGTCTGC 64 2568 1104346 N/A N/A 11381 11400 CTGCTGGAGTAAGATGAGCT 99 2569 1104362 N/A N/A 11478 11497 TTCTTGATAGTAACCACAGC 53 2570 1104378 N/A N/A 11558 11577 TAGGAGTTCACACAGACCCC 164  2571

TABLE 35 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  8 517 1103163 233 252 3681 3700 GAAGCCAGCATTGAGTGCCC 109  2572 1103179 484 503 3932 3951 TGTGCCAGATTGTCCCTCTC  13* 2573 1103195 713 732 5599 5618 CACATGGACCTGCTGTCGGG 107  2574 1103211 951 970 7308 7327 TGCCGCGCAGAGACTCCAGG 90 2575 1103227 1317 1336 N/A N/A ACTCCTTAATGACCTCTCCA 151  2576 1103243 1434 1453 11723 11742 GAAATGTGCCAGCAGAGGCG 96 2577 1103259 1581 1600 11870 11889 GGGTGAGTTTCTTGTTAGTT 15 2578 1103275 1712 1731 12001 12020 AGTTTCCATAACAACAGGAA 91 2579 1103291 1851 1870 12140 12159 AAAACAAGCCTCTGGCCAGG 115  2580 1103307 1881 1900 12170 12189 GGATATCCCACCTCATAAAA 107  2581 1103323 2030 2049 12319 12338 CACCCATCTTAGACTGATCA 116  2582 1103339 2197 2216 12486 12505 CACAACAGATCCCCCAAGTG 110  2583 1103355 2409 2428 12698 12717 AATTCCCACAATCCAGAGGC 72 2584 1103371 2455 2474 12744 12763 CTTTCTCTCCTGTTTCAGCA 20 2585 1103387 2506 2525 12795 12814 AGGCCTTTAGAAATGGGACA 76 2586 1103403 2656 2675 12945 12964 GGACCGCAAGAGGCCCTTGG 83 2587 1103419 2707 2726 12996 13015 AAATAGGGCACTACCTAGAA 100  2588 1103435 2831 2850 13120 13139 CTGCTCAGTCAAAGCAGAGT 99 2589 1103467 N/A N/A 8765 8784 TCTTGTGACCTTGTGATTTT 55 2590 1103483 N/A N/A 8894 8913 TCACTCCCTGTCAAGCTGGG 130  2591 1103499 N/A N/A 9029 9048 TTAATGTACAGTTACTCTGT 98 2592 1103515 N/A N/A 9214 9233 CGATGGAGCCTCAGGGATGA 57 2593 1103531 N/A N/A 9253 9272 AGGGAAGAATCCTCTGAACT 59 2594 1103547 N/A N/A 9298 9317 AGCAGGGAACATAAAACTTT 79 2595 1103563 N/A N/A 8458 8477 TGATCCTCAGTCCCAGTCTG 95 2596 1103579 N/A N/A 8552 8571 AAGCCCCACCTAGAAGTACC 131  2597 1103595 N/A N/A 3965 3984 CCTCCTCACTTCTGCCTCAC  77* 2598 1103611 N/A N/A 4046 4065 TCGGCCCCTCCCTGAGACTT 74 2599 1103627 N/A N/A 4220 4239 TCCTGCACTGCTTTCCCCAG 80 2600 1103643 N/A N/A 4385 4404 TGCTCTTTCCCTCACTTTCT 119  2601 1103659 N/A N/A 4484 4503 ATTATGACCACCGCTTCACA 54 2602 1103675 N/A N/A 4603 4622 ACACACGCACATGTCCTCCT 157  2603 1103691 N/A N/A 4751 4770 CCTTAGACAGAGGACTTGTC 90 2604 1103707 N/A N/A 4967 4986 CCACCTCCCTGACCTGTCTA 102* 2605 1103723 N/A N/A 5301 5320 AGTTGCAATCTCTGTGTTGA 105  2606 1103739 N/A N/A 5391 5410 TCCTGTCTCTACCTGCCAAT 81 2607 1103755 N/A N/A 5457 5476 CCAGGAGTTCGAATGCTCTC 97 2608 1103771 N/A N/A 5557 5576 AACCTCCTGACCAGGGTGAG 87 2609 1103787 N/A N/A 5839 5858 TCTGGTGAAAGTCAGTCACC 99 2610 1103803 N/A N/A 5910 5929 TAGCAGTAATAATAATGGGT 53 2611 1103819 N/A N/A 5967 5986 TGAAAAGCACAGGGCTTAGA 118  2612 1103835 N/A N/A 6016 6035 CTCTCTCATCTGTCAAAGAA 64 2613 1103851 N/A N/A 6164 6183 ATATGGTAGAGGCTCAGTAA 70 2614 1103867 N/A N/A 6253 6272 GGTGTTCTCTACGGGCACTA 87 2615 1103883 N/A N/A 6289 6308 TCCTGGCTCCCACACTACAT 109  2616 1103899 N/A N/A 6402 6421 GTGCTTTTCTGCCTCCAGGC 32 2617 1103915 N/A N/A 6489 6508 CCCTGCTCAGACACCAGTGG 157  2618 1103931 N/A N/A 6899 6918 GAGCGGAGGCCTGGGTGTTT 123  2619 1103947 N/A N/A 7005 7024 TAGCACAACACCTGGTCAGC 92 2620 1103963 N/A N/A 7127 7146 GGAAATCAGGGAGGTGAGCA 58 2621 1103979 N/A N/A 7385 7404 GCCCTTCTCCCCTGGCATCT 83 2622 1103995 N/A N/A 7799 7818 CTCTACCGTGAGGCAGCAGG 97 2623 1104011 N/A N/A 7909 7928 CTAGTGACTGCCTGCTATGT 112  2624 1104027 N/A N/A 8007 8026 GGGAGTATGCCTCTTAGTTT 55 2625 1104043 N/A N/A 8071 8090 AGAAAGTTCCAAGGAGGCAG 112  2626 1104059 N/A N/A 8208 8227 AACTCCATCTCCTAGCTTTT 85 2627 1104075 N/A N/A 9348 9367 CCGAGAGAGAAAAATATAAC 66 2628 1104091 N/A N/A 9404 9423 ATTTCAGGGCCAATGCAAGT 85 2629 1104107 N/A N/A 9448 9467 TTCTAGTCCAGAAGAAGAGG 72 2630 1104123 N/A N/A 9495 9514 GATGCTGAATTAAGTCCTGA 41 2631 1104139 N/A N/A 9527 9546 TACCTCTACTAGTCAGCCTG 78 2632 1104155 N/A N/A 9554 9573 CTGTGACTATCTAGGATTTG 70 2633 1104171 N/A N/A 9573 9592 TTATCTGTGCTTTAGTGACC 54 2634 1104187 N/A N/A 9642 9661 CATATTAGTGCTCAATACAC 59 2635 1104203 N/A N/A 9703 9722 ACTTCTCTAGGTGGGAGAGA 58 2636 1104219 N/A N/A 10136 10155 GTAAGACTCCGTCGAAAGCA 99 2637 1104235 N/A N/A 10573 10592 TTTCTGAAAACCCAGCACGG 132  2638 1104251 N/A N/A 10730 10749 TGAGAACCTATGCAACCGAG 62 2639 1104267 N/A N/A 10798 10817 TTTCAGGGCATGAGCCATCC 140  2640 1104283 N/A N/A 11070 11089 TCAGTTTCCTTGCTCTCCTC 82 2641 1104299 N/A N/A 11146 11165 GATCCCATCTAGTGGCTTTG 39 2642 1104315 N/A N/A 11213 11232 TTCTCTTTCTCTCCCTGGCA 85 2643 1104331 N/A N/A 11265 11284 CCCATCCCCAACTGTGTCTG 73 2644 1104347 N/A N/A 11384 11403 AAGCTGCTGGAGTAAGATGA 122  2645 1104363 N/A N/A 11483 11502 TACTTTTCTTGATAGTAACC 74 2646 1104379 N/A N/A 11559 11578 TTAGGAGTTCACACAGACCC 85 2647

TABLE 36 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG 10 517 1103164 254 273 3702 3721 CTCACTGGCCCGGGTCTCCT 133  2648 1103180 485 504 3933 3952 CTGTGCCAGATTGTCCCTCT  16* 2649 1103196 714 733 5600 5619 CCACATGGACCTGCTGTCGG 121  2650 1103212 1046 1065 7618 7637 TTCCTCCAGCCGCGCCAGCG 135  2651 1103228 1324 1343 11613 11632 TGCTTGGACTCCTTAATGAC 97 2652 1103244 1436 1455 11725 11744 GGGAAATGTGCCAGCAGAGG 39 2653 1103260 1601 1620 11890 11909 CCCTCCAGACTGCCCCTTGG 95 2654 1103276 1726 1745 12015 12034 CCATCTCTGGCAACAGTTTC 71 2655 1103292 1856 1875 12145 12164 AAGACAAAACAAGCCTCTGG 88 2656 1103308 1882 1901 12171 12190 GGGATATCCCACCTCATAAA 88 2657 1103324 2031 2050 12320 12339 CCACCCATCTTAGACTGATC 100  2658 1103340 2198 2217 12487 12506 ACACAACAGATCCCCCAAGT 90 2659 1103356 2410 2429 12699 12718 TAATTCCCACAATCCAGAGG 115  2660 1103372 2459 2478 12748 12767 TCCCCTTTCTCTCCTGTTTC 101  2661 1103388 2508 2527 12797 12816 AGAGGCCTTTAGAAATGGGA 102  2662 1103404 2662 2681 12951 12970 AAGAAGGGACCGCAAGAGGC 83 2663 1103420 2715 2734 13004 13023 CAATTGTAAAATAGGGCACT 115  2664 1103436 2835 2854 13124 13143 CAGTCTGCTCAGTCAAAGCA 121  2665 1103468 N/A N/A 8766 8785 ATCTTGTGACCTTGTGATTT 75 2666 1103484 N/A N/A 8895 8914 CTCACTCCCTGTCAAGCTGG 98 2667 1103500 N/A N/A 9033 9052 CAGTTTAATGTACAGTTACT 90 2668 1103516 N/A N/A 9215 9234 GCGATGGAGCCTCAGGGATG 28 2669 1103532 N/A N/A 9254 9273 GAGGGAAGAATCCTCTGAAC 86 2670 1103548 N/A N/A 9305 9324 CATTAAGAGCAGGGAACATA 85 2671 1103564 N/A N/A 8492 8511 GAAGGCCCCCAGGGAGAGCT 72 2672 1103580 N/A N/A 8553 8572 CAAGCCCCACCTAGAAGTAC 107  2673 1103596 N/A N/A 3966 3985 CCCTCCTCACTTCTGCCTCA  98* 2674 1103612 N/A N/A 4047 4066 TTCGGCCCCTCCCTGAGACT 77 2675 1103628 N/A N/A 4227 4246 CCGCTGCTCCTGCACTGCTT 123  2676 1103644 N/A N/A 4387 4406 CCTGCTCTTTCCCTCACTTT 110  2677 1103660 N/A N/A 4485 4504 TATTATGACCACCGCTTCAC 85 2678 1103676 N/A N/A 4604 4623 CACACACGCACATGTCCTCC 106  2679 1103692 N/A N/A 4754 4773 GGGCCTTAGACAGAGGACTT 97 2680 1103708 N/A N/A 4970 4989 CCTCCACCTCCCTGACCTGT  91* 2681 1103724 N/A N/A 5302 5321 CAGTTGCAATCTCTGTGTTG 127  2682 1103740 N/A N/A 5392 5411 TTCCTGTCTCTACCTGCCAA 153  2683 1103756 N/A N/A 5475 5494 CTTCTGCCTGCCCCTCGGCC 109  2684 1103772 N/A N/A 5558 5577 GAACCTCCTGACCAGGGTGA 116  2685 1103788 N/A N/A 5840 5859 CTCTGGTGAAAGTCAGTCAC 112  2686 1103804 N/A N/A 5911 5930 GTAGCAGTAATAATAATGGG 93 2687 1103820 N/A N/A 5969 5988 CATGAAAAGCACAGGGCTTA 94 2688 1103836 N/A N/A 6017 6036 GCTCTCTCATCTGTCAAAGA 59 2689 1103852 N/A N/A 6197 6216 AGACACCTCTCTGTGTCCTG 60 2690 1103868 N/A N/A 6255 6274 GTGGTGTTCTCTACGGGCAC 95 2691 1103884 N/A N/A 6308 6327 GCCCCCTCTACAGTGTCTTT 85 2692 1103900 N/A N/A 6405 6424 TCTGTGCTTTTCTGCCTCCA 59 2693 1103916 N/A N/A 6493 6512 CTCACCCTGCTCAGACACCA 105  2694 1103932 N/A N/A 6900 6919 CGAGCGGAGGCCTGGGTGTT 59 2695 1103948 N/A N/A 7006 7025 CTAGCACAACACCTGGTCAG 106  2696 1103964 N/A N/A 7195 7214 AGGTCTGCAAACTAGGTGGG 93 2697 1103980 N/A N/A 7414 7433 CCCGCCCTCGACCCAGGTCC 121  2698 1103996 N/A N/A 7802 7821 GAGCTCTACCGTGAGGCAGC 101  2699 1104012 N/A N/A 7911 7930 ATCTAGTGACTGCCTGCTAT 95 2700 1104028 N/A N/A 8031 8050 AGTCCAATCTTGGCTGGGAA 80 2701 1104044 N/A N/A 8073 8092 GAAGAAAGTTCCAAGGAGGC 94 2702 1104060 N/A N/A 8209 8228 TAACTCCATCTCCTAGCTTT 89 2703 1104076 N/A N/A 9349 9368 CCCGAGAGAGAAAAATATAA 99 2704 1104092 N/A N/A 9406 9425 TCATTTCAGGGCCAATGCAA 63 2705 1104108 N/A N/A 9450 9469 TATTCTAGTCCAGAAGAAGA 127  2706 1104124 N/A N/A 9504 9523 AGCCTTTCTGATGCTGAATT 52 2707 1104140 N/A N/A 9528 9547 TTACCTCTACTAGTCAGCCT 75 2708 1104156 N/A N/A 9555 9574 CCTGTGACTATCTAGGATTT 52 2709 1104172 N/A N/A 9574 9593 TTTATCTGTGCTTTAGTGAC 50 2710 1104188 N/A N/A 9651 9670 CATGTGGCACATATTAGTGC 97 2711 1104204 N/A N/A 9704 9723 TACTTCTCTAGGTGGGAGAG 83 2712 1104220 N/A N/A 10137 10156 AGTAAGACTCCGTCGAAAGC 124  2713 1104236 N/A N/A 10574 10593 CTTTCTGAAAACCCAGCACG 87 2714 1104252 N/A N/A 10731 10750 ATGAGAACCTATGCAACCGA 68 2715 1104268 N/A N/A 10807 10826 CTCATGGACTTTCAGGGCAT 84 2716 1104284 N/A N/A 11076 11095 TTACATTCAGTTTCCTTGCT 108  2717 1104300 N/A N/A 11147 11166 GGATCCCATCTAGTGGCTTT 94 2718 1104316 N/A N/A 11222 11241 TAACTTTAATTCTCTTTCTC 135  2719 1104332 N/A N/A 11266 11285 CCCCATCCCCAACTGTGTCT 80 2720 1104348 N/A N/A 11385 11404 TAAGCTGCTGGAGTAAGATG 92 2721 1104364 N/A N/A 11484 11503 TTACTTTTCTTGATAGTAAC 89 2722 1104380 N/A N/A 11573 11592 GCACAGTGCAACAGTTAGGA 38 2723

TABLE 37 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 NO: 2 NO: 2 SEQ Compound Start Stop Start Stop GFAP ID No. Site Site Site Site Sequence (5′ to 3′) (% UTC) NO 1047582 3047 3066 13336 13355 GTCTTTATTTTTCCTCAGCG  6 517 1103165 261 280 3709 3728 CTGCCCGCTCACTGGCCCGG 98 2724 1103181 505 524 3953 3972 TGCCTCACAGTGGCCAGGTC  45* 2725 1103197 756 775 5642 5661 CTTTCAGGGCTGCGGTGAGG 99 2726 1103213 1048 1067 7620 7639 TCTTCCTCCAGCCGCGCCAG 53 2727 1103229 1325 1344 11614 11633 CTGCTTGGACTCCTTAATGA 73 2728 1103245 1497 1516 11786 11805 AAGCTGACCTAGGGACAGAG 106  2729 1103261 1602 1621 11891 11910 CCCCTCCAGACTGCCCCTTG 73 2730 1103277 1727 1746 12016 12035 TCCATCTCTGGCAACAGTTT 48 2731 1103293 1857 1876 12146 12165 AAAGACAAAACAAGCCTCTG 71 2732 1103309 1883 1902 12172 12191 AGGGATATCCCACCTCATAA 159  2733 1103325 2057 2076 12346 12365 TGACTGCCCCAGGTGGCAGG 116  2734 1103341 2199 2218 12488 12507 TACACAACAGATCCCCCAAG 86 2735 1103357 2411 2430 12700 12719 TTAATTCCCACAATCCAGAG 104  2736 1103373 2460 2479 12749 12768 ATCCCCTTTCTCTCCTGTTT 76 2737 1103389 2509 2528 12798 12817 AAGAGGCCTTTAGAAATGGG 71 2738 1103405 2663 2682 12952 12971 TAAGAAGGGACCGCAAGAGG 81 2739 1103421 2716 2735 13005 13024 ACAATTGTAAAATAGGGCAC 81 2740 1103437 2837 2856 13126 13145 ACCAGTCTGCTCAGTCAAAG 90 2741 1103469 N/A N/A 8767 8786 TATCTTGTGACCTTGTGATT 54 2742 1103485 N/A N/A 8896 8915 GCTCACTCCCTGTCAAGCTG 76 2743 1103501 N/A N/A 9042 9061 AAGCTCTGCCAGTTTAATGT 57 2744 1103517 N/A N/A 9216 9235 AGCGATGGAGCCTCAGGGAT 83 2745 1103533 N/A N/A 9255 9274 TGAGGGAAGAATCCTCTGAA 92 2746 1103549 N/A N/A 9306 9325 ACATTAAGAGCAGGGAACAT 105  2747 1103565 N/A N/A 8499 8518 GTGTCACGAAGGCCCCCAGG 72 2748 1103581 N/A N/A 8556 8575 CTGCAAGCCCCACCTAGAAG 94 2749 1103597 N/A N/A 3989 4008 TCACAAGGCCCCCCTTCCCC 98 2750 1103613 N/A N/A 4048 4067 GTTCGGCCCCTCCCTGAGAC 90 2751 1103629 N/A N/A 4228 4247 CCCGCTGCTCCTGCACTGCT 83 2752 1103645 N/A N/A 4389 4408 TGCCTGCTCTTTCCCTCACT 88 2753 1103661 N/A N/A 4489 4508 ATTTTATTATGACCACCGCT 97 2754 1103677 N/A N/A 4625 4644 TCACTGTTGCACACACACAC 124  2755 1103693 N/A N/A 4775 4794 GCAGGAGGATTAAGGGTTGG 76 2756 1103709 N/A N/A 5010 5029 TGGGTGGCCATCAATCCTTT 97 2757 1103725 N/A N/A 5303 5322 TCAGTTGCAATCTCTGTGTT 65 2758 1103741 N/A N/A 5394 5413 ATTTCCTGTCTCTACCTGCC 95 2759 1103757 N/A N/A 5480 5499 GTCCTCTTCTGCCTGCCCCT 90 2760 1103773 N/A N/A 5722 5741 CAGGGCTACCTTGGAGCGGT 71 2761 1103789 N/A N/A 5848 5867 TCTCACTTCTCTGGTGAAAG 82 2762 1103805 N/A N/A 5912 5931 AGTAGCAGTAATAATAATGG 86 2763 1103821 N/A N/A 5973 5992 AATCCATGAAAAGCACAGGG 88 2764 1103837 N/A N/A 6018 6037 GGCTCTCTCATCTGTCAAAG 24 2765 1103853 N/A N/A 6198 6217 CAGACACCTCTCTGTGTCCT 108  2766 1103869 N/A N/A 6256 6275 TGTGGTGTTCTCTACGGGCA 62 2767 1103885 N/A N/A 6312 6331 AAATGCCCCCTCTACAGTGT 110  2768 1103901 N/A N/A 6406 6425 CTCTGTGCTTTTCTGCCTCC 67 2769 1103917 N/A N/A 6500 6519 TCGGGCCCTCACCCTGCTCA 92 2770 1103933 N/A N/A 6902 6921 GGCGAGCGGAGGCCTGGGTG 12 2771 1103949 N/A N/A 7016 7035 ACCTCAGCACCTAGCACAAC 90 2772 1103965 N/A N/A 7197 7216 TCAGGTCTGCAAACTAGGTG 72 2773 1103981 N/A N/A 7415 7434 CCCCGCCCTCGACCCAGGTC 131  2774 1103997 N/A N/A 7804 7823 ATGAGCTCTACCGTGAGGCA 88 2775 1104013 N/A N/A 7913 7932 ACATCTAGTGACTGCCTGCT 94 2776 1104029 N/A N/A 8034 8053 AGGAGTCCAATCTTGGCTGG 74 2777 1104045 N/A N/A 8074 8093 TGAAGAAAGTTCCAAGGAGG 95 2778 1104061 N/A N/A 8215 8234 AAAGTCTAACTCCATCTCCT 131  2779 1104077 N/A N/A 9358 9377 CCCGCCCCGCCCGAGAGAGA 83 2780 1104093 N/A N/A 9408 9427 AATCATTTCAGGGCCAATGC 24 2781 1104109 N/A N/A 9451 9470 ATATTCTAGTCCAGAAGAAG 123  2782 1104125 N/A N/A 9505 9524 TAGCCTTTCTGATGCTGAAT 57 2783 1104141 N/A N/A 9529 9548 ATTACCTCTACTAGTCAGCC 59 2784 1104157 N/A N/A 9556 9575 ACCTGTGACTATCTAGGATT 82 2785 1104173 N/A N/A 9575 9594 ATTTATCTGTGCTTTAGTGA 41 2786 1104189 N/A N/A 9652 9671 ACATGTGGCACATATTAGTG 98 2787 1104205 N/A N/A 9705 9724 CTACTTCTCTAGGTGGGAGA 79 2788 1104221 N/A N/A 10138 10157 GAGTAAGACTCCGTCGAAAG 89 2789 1104237 N/A N/A 10608 10627 TCAGCCCCTCTGCAAGCCCT 85 2790 1104253 N/A N/A 10732 10751 TATGAGAACCTATGCAACCG 72 2791 1104269 N/A N/A 10808 10827 TCTCATGGACTTTCAGGGCA 112  2792 1104285 N/A N/A 11077 11096 ATTACATTCAGTTTCCTTGC 103  2793 1104301 N/A N/A 11148 11167 AGGATCCCATCTAGTGGCTT 91 2794 1104317 N/A N/A 11224 11243 ACTAACTTTAATTCTCTTTC 96 2795 1104333 N/A N/A 11274 11293 AAAGCCCTCCCCATCCCCAA 128  2796 1104349 N/A N/A 11387 11406 GGTAAGCTGCTGGAGTAAGA 26 2797 1104365 N/A N/A 11485 11504 CTTACTTTTCTTGATAGTAA 36 2798 1104381 N/A N/A 11605 11624 CTCCTTAATGACCTGCAGGG 74 2799

TABLE 38 Reduction of GFAP RNA by 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages in U251 cells SEQ SEQ ID ID NO: 3 NO: 3 SEQ Compound Start Stop GFAP ID Number Site Site Sequence (5′ to 3 ) (% UTC) NO 1103451 1220 1239 CCCTCGAATCTGCAGGTTGG 94 2800 1103452 1227 1246 TTTTGCCCCCTCGAATCTGC 106 2801 1103453 1228 1247 CTTTTGCCCCCTCGAATCTG 84 2802 1103454 1229 1248 GCTTTTGCCCCCTCGAATCT 95 2803 1103455 1231 1250 GTGCTTTTGCCCCCTCGAAT 59 2804 1103456 1233 1252 TGGTGCTTTTGCCCCCTCGA 92 2805 1103457 1235 1254 TTTGGTGCTTTTGCCCCCTC 60 2806 1103554 1813 1832 TTAATATTTAACATTAAGAG 77 2807 1047595 1222 1241 CCCCCTCGAATCTGCAGGTT 109 284 1047596 1223 1242 GCCCCCTCGAATCTGCAGGT 107 362 1047597 1225 1244 TTGCCCCCTCGAATCTGCAG 65 440 1047598 1230 1249 TGCTTTTGCCCCCTCGAATC 115 518 1047599 1232 1251 GGTGCTTTTGCCCCCTCGAA 43 596 1047600 1234 1253 TTGGTGCTTTTGCCCCCTCG 86 674

Example 3: Effect of Modified Oligonucleotides on Human GFAP RNA In Vitro, Multiple Doses

Modified oligonucleotides selected from the examples above were tested at various doses in U251 cells. Cultured U251 cells at a density of 10,000 cells per well were treated using free uptake with various concentrations of modified oligonucleotide as specified in the tables below. After a treatment period of approximately 48 hours, total RNA was isolated from the cells and GFAP RNA levels were measured by quantitative real-time RTPCR. Human GFAP primer probe set RTS37485 was used to measure RNA levels, as described above. GFAP RNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent of GFAP RNA, relative to untreated control cells (% control). Where possible, the half maximal inhibitory concentration (IC₅₀) of each modified oligonucleotide was calculated using a linear regression on a log/linear plot of the data in Excel. In some cases, an IC₅₀ could not be reliably calculated and the data point is marked as “NC”. Modified oligonucleotides marked with an asterisk (*) indicate that the modified oligonucleotide is complementary to the amplicon region of the primer probe set. Additional assays may be used to measure the potency and efficacy of the modified oligonucleotides complementary to the amplicon region.

TABLE 39 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) IC₅₀ Compound No. 125 nM 500 nM 2000 nM 8000 nM (μM) 1047225 76 57 47 42 2.0 1047257 60 51 39 33 0.5 1047258 51 50 38 29 0.2 1047386 99 82 41 26 1.9 1047387 109 73 33 13 1.3 1047448 85 122 99 94 NC 1047466 107 82 77 46 7.8 1047497 120 117 107 136 NC 1047512 116 112 97 87 NC 1047584 94 54 23 10 0.8 1047608 125 133 79 52 NC 1047609 88 82 57 33 3.0 1047610 92 75 43 19 1.5 1047913 98 72 49 25 1.9 1048203 120 95 57 31 3.3 1048267 99 100 93 70 NC 1048296 99 91 73 72 NC 1048344 77 97 88 86 NC 1048361 92 115 85 109 NC

TABLE 40 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) IC₅₀ Compound No. 125 nM 500 nM 2000 nM 8000 nM (μM) 1047325 81 86 76 67 NC 1047357 74 63 47 22 1.1 1047372 145 143 125 99 NC 1047373 105 73 43 21 1.7 1047374 150 122 84 46 7.3 1047388 108 116 62 32 4.2 1047500 100 105 74 60 NC 1047515 93 95 81 65 NC 1047518 125 99 81 51 NC 1047579 92 113 81 53 NC 1047580 96 88 58 41 4.3 1047581 73 60 32 17 0.7 1047582 72 29 12 7 0.3 1047584 100 60 23 11 0.6 1047613 107 107 79 44 NC 1047662 99 86 83 47 NC 1047707 100 126 91 75 NC 1048027 82 83 59 56 NC 1048350 104 85 57 36 3.4

TABLE 41 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) IC₅₀ Compound No. 125 nM 500 nM 2000 nM 8000 nM (μM) 1047235 80 81 68 57 NC 1047328 91 79 55 39 3.5 1047362 110 77 52 20 2.0 1047391 87 79 61 45 5.5 1047394 78 74 52 36 2.6 1047503 92 94 80 59 NC 1047522 81 81 63 50 NC 1047583 97 54 19 12 0.8 1047584 81 50 19 10 0.6 1047585 85 73 38 18 1.2 1047586 75 64 41 23 1.1 1047587 92 66 41 19 1.3 1047599 92 94 83 78 NC 1047601 103 89 68 50 NC 1047762 99 84 70 71 NC 1047955 95 104 86 92 NC 1048018 85 84 74 51 NC 1048351 98 97 75 59 NC 1048355 114 114 90 61 NC

TABLE 42 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) IC₅₀ Compound No. 125 nM 500 nM 2000 nM 8000 nM (μM) 1047429 80 58 32 15 0.8 1047444 80 98 83 73 NC 1047492 92 99 79 58 NC 1047573 80 74 59 44 4.7 1047584 87 53 20 8 0.7 1047588 69 49 24 14 0.5 1047589 101 79 47 24 2.0 1047590 70 55 35 24 0.7 1047591 92 67 34 21 1.2 1047734 86 75 69 60 NC 1047990 75 75 58 40 4.1 1048055 103 96 61 42 5.0 1048151 94 76 23 16 1.1 1048182 85 76 56 35 2.9 1048195 105 104 87 87 NC 1048196 94 91 73 62 NC 1048228 91 81 87 93 NC 1048341 80 71 61 46 6.1 1048372 94 88 84 59 NC

TABLE 43 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) IC₅₀ Compound No. 125 nM 500 nM 2000 nM 8000 nM (μM) 1047321 94 91 84 79 NC 1047337 94 89 68 61 NC 1047368 86 82 79 81 NC 1047385 86 80 63 58 NC 1047432 89 84 75 89 NC 1047465 82 67 62 49 7.3 1047469 104 95 73 58 NC 1047513 106 100 96 80 NC 1047576 82 83 63 49 NC 1047584 88 63 31 14 1.0 1047625 86 80 73 55 NC 1047675 101 88 92 82 NC 1047688 99 100 96 100 NC 1047705 100 92 78 68 NC 1047866 90 81 71 78 NC 1048125 103 91 86 84 NC 1048187 100 88 82 74 NC 1048188 99 64 80 81 NC 1048201 80 65 41 23 1.2

TABLE 44 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) IC₅₀ Compound No. 125 nM 500 nM 2000 nM 8000 nM (μM) 1047298 87 83 74 75 NC 1047474 102 93 94 87 NC 1047523 88 76 61 42 4.6 1047582 79 37 18 12 0.4 1047584 100 72 43 20 1.6 1047607 92 85 68 56 NC 1047717 106 110 92 80 NC 1047746 108 110 106 110 NC 1047790 91 86 81 70 NC 1047799 104 100 95 93 NC 1047840 92 88 75 58 NC 1047859 96 87 87 78 NC 1047886 84 79 68 68 NC 1047907 97 90 80 71 NC 1048093 97 89 93 90 NC 1048144 101 93 99 108 NC 1048207 97 82 76 74 NC 1048223 91 77 77 68 NC 1048350 104 80 57 44 4.4

TABLE 45 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 25 11 7 5 NC 1072814 62 45 22 14 0.7 1072815 86 57 42 28 2.3 1072818 78 54 33 16 1.5 1072834 33 22 13 8 NC 1072835 54 30 15 9 NC 1072855 64 44 27 16 0.8 1072862 59 40 20 9 0.6 1072863 83 54 35 20 1.7 1072868 80 50 23 9 1.2 1072872 21 13 9 7 NC 1072886 88 60 42 21 2.2 1072986 84 68 22 11 1.7 1073003 93 79 71 52 NC 1073034 97 80 30 30 2.9 1073035 78 80 41 27 2.8 1073063 53 29 15 7 NC 1073106 87 76 57 39 5.2 1073107 79 65 58 33 3.7

TABLE 46 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 36 15 8 6 NC 1072813 69 58 36 17 1.4 1072824 85 63 38 23 2.2 1072849 66 32 21 14 0.6 1072853 116 98 70 24 5.1 1072856 90 57 32 14 1.8 1072857 66 45 32 17 1.0 1072861 68 45 28 16 1.0 1072864 87 62 43 22 2.3 1072973 101 74 40 13 2.5 1072980 39 45 27 16 NC 1073009 87 75 83 35 8.2 1073033 87 44 19 24 1.4 1073045 60 43 24 12 0.7 1073060 68 43 28 16 0.9 1073064 61 41 31 19 0.7 1073065 69 61 36 18 1.5 1073077 95 70 44 23 2.8 1073093 57 33 20 10 0.5

TABLE 47 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 70 30 9 5 0.6 1103198 91 86 82 73 NC 1103246 102 84 81 62 NC 1103278 89 96 72 51 NC 1103279 86 69 43 30 2.9 1103359 82 75 55 39 5.0 1103470 86 88 73 55 NC 1103471 68 63 45 36 2.6 1103502 104 93 75 51 NC 1103567 88 66 45 23 2.5 1104014 91 91 56 56 NC 1104078 92 100 103 92 NC 1104127 67 52 42 33 1.6 1104144 93 63 34 16 2.1 1104158 50 51 55 23 1.0 1104159 65 59 40 25 1.6 1104174 91 93 63 43 7.9 1104175 69 46 23 13 0.9 1104191 84 76 52 34 4.0

TABLE 48 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 66 30 11 6 0.6 1103202 79 79 54 43 6.0 1103217 77 38 16 5 0.9 1103218 92 74 50 33 3.7 1103330 84 79 56 51 NC 1103345 112 125 111 149 NC 1103377 115 134 142 151 NC 1103409 82 93 70 45 NC 1103570 79 64 43 24 2.3 1103571 101 74 61 34 4.6 1103872 93 79 28 21 2.4 1103970 88 78 75 74 NC 1104096 109 112 65 47 9.2 1104131 87 79 54 44 6.1 1104145 99 89 65 39 6.7 1104161 102 90 75 52 NC 1104178 72 67 57 53 NC 1104225 87 82 57 39 5.4 1104307 97 87 57 32 4.7

TABLE 49 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 58 29 10 7 0.4 1103236 81 62 27 12 1.6 1103253 68 56 36 37 1.8 1103268 86 62 37 30 2.4 1103285 91 79 56 29 4.0 1103300 100 98 83 81 NC 1103365 83 86 64 42 8.2 1103428 87 73 50 26 3.2 1103491 86 70 70 55 NC 1103620 90 62 65 73 NC 1104037 77 92 66 38 7.8 1104116 85 63 42 23 2.3 1104118 99 74 38 20 2.6 1104132 107 79 53 35 4.5 1104133 70 67 43 29 2.3 1104165 91 86 59 32 4.8 1104276 94 71 18 15 1.9 1104309 73 60 36 20 1.6 1104310 82 57 30 13 1.6

TABLE 50 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 69 30 11 7 0.6 1103241 87 71 60 50 NC 1103257 75 83 47 26 3.1 1103258 81 70 44 23 2.4 1103272 94 91 52 26 4.0 1103369 88 78 59 38 5.4 1103370 83 88 59 44 8.0 1103400 95 101 87 76 NC 1103448 91 73 49 32 3.6 1103465 88 68 47 31 3.1 1103592 96 111 75 48 NC  1103722* 21 24 21 19 NC 1103816 108 118 95 76 NC 1104072 111 83 63 17 3.8 1104122 84 73 65 48 NC 1104153 78 83 55 50 NC 1104280 111 84 55 25 3.9 1104312 91 71 52 32 3.7 1104361 87 81 65 42 7.4

TABLE 51 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 60 36 12 8 0.5 1103244 99 79 55 42 5.7 1103259 85 70 42 26 2.6 1103322 87 82 66 46 9.5 1103371 86 97 64 38 7.3 1103516 104 123 90 69 NC 1103837 82 62 40 26 2.2 1103899 94 87 68 48 NC 1103932 74 83 68 64 NC 1103933 79 63 11 9 1.3 1104093 90 61 33 24 2.1 1104123 91 114 81 61 NC 1104156 83 86 71 55 NC 1104173 87 89 60 42 7.2 1104298 105 102 80 57 NC 1104299 101 109 84 72 NC 1104349 104 71 57 35 4.3 1104365 95 75 63 40 5.8 1104380 95 83 61 45 7.3

TABLE 52 Dose-dependent percent of human GFAP RNA compared to untreated control in U251 cells by modified oligonucleotides GFAP RNA (% control) Compound No. 370 nM 1111 nM 3333 nM 10000 nM IC50 (μM) 1047582 63 26 10 6 0.5 1103254 93 85 62 37 5.8 1103255 59 49 23 14 0.8 1103256 84 54 22 10 1.4 1103366 105 79 58 58 NC 1103368 92 79 44 21 2.9 1103430 97 86 60 42 6.3 1103446 84 79 51 36 4.3 1103447 88 79 49 32 3.7 1103478 90 95 62 46 9.1 1103558 82 92 59 32 5.2 1103591 73 69 51 31 3.0 1104038 68 51 31 36 1.4 1104039 80 68 43 36 3.1 1104151 80 65 46 28 2.6 1104152 94 71 47 22 2.8 1104166 94 70 45 27 3.0 1104311 70 60 40 22 1.7 1104326 97 85 64 52 NC

Example 4: Tolerability of Modified Oligonucleotides Complementary to Human GFAP in Wild-Type Mice, 3 Hour Study

Modified oligonucleotides described above were tested in wild-type female C57/B16 mice to assess the tolerability of the oligonucleotides. Wild-type female C57/B16 mice each received a single ICV dose of 700 μg of modified oligonucleotide listed in the table below. Each treatment group consisted of 4 mice. A group of 4 mice received PBS as a negative control for each experiment (identified in separate tables below). At 3 hours post-injection, mice were evaluated according to seven different criteria. The criteria are (1) the mouse was bright, alert, and responsive; (2) the mouse was standing or hunched without stimuli; (3) the mouse showed any movement without stimuli; (4) the mouse demonstrated forward movement after it was lifted; (5) the mouse demonstrated any movement after it was lifted; (6) the mouse responded to tail pinching; (7) regular breathing. For each of the 7 criteria, a mouse was given a subscore of 0 if it met the criteria and 1 if it did not (the functional observational battery score or FOB). After all 7 criteria were evaluated, the scores were summed for each mouse and averaged within each treatment group. The results are presented in the tables below.

TABLE 53 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1047198 3 1047258 3 1047328 7 1047362 0 1047373 0 1047386 4 1047387 0 1047388 2 1047391 5 1047580 0 1047582 5 1047583 2 1047584 1 1047585 0 1047586 0 1047587 0 1047588 0 1047589 1 1047590 2 1047591 4 1047610 5 1048203 5 1048267 6 1048350 4

TABLE 54 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1047257 5 1047357 7 1047374 5 1047394 6 1047429 0 1047444 2 1047448 0 1047492 2 1047497 2 1047500 1 1047518 5 1047573 1 1047581 1 1047599 1 1047601 6 1047608 2 1047609 5 1047913 1 1047990 2 1048027 2 1048151 4 1048182 0 1048296 3 1048361 0

TABLE 55 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1047211 2 1047223 0 1047225 1 1047298 0 1047306 0 1047316 2 1047352 4 1047353 2 1047402 2 1047432 0 1047522 0 1047523 2 1047532 2 1047579 2 1047598 4 1047602 5 1047662 4 1047679 2 1047711 2 1047811 0 1047884 3 1048201 1 1048204 0 1048227 1

TABLE 56 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1072813 0 1072814 1 1072818 4 1072834 4 1072835 0 1072849 5 1072855 4 1072856 6 1072857 4 1072861 6 1072862 4 1072863 3 1072868 7 1072872 5 1072886 1 1072980 1 1072986 6 1073033 5 1073045 6 1073060 6 1073063 6 1073064 6 1073065 6 1073093 6

TABLE 57 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1103217 5 1103368 3 1103370 3 1103371 3 1103465 6 1103502 5 1103516 7 1103567 5 1103837 4 1103899 2 1104093 5 1104118 6 1104133 6 1104145 2 1104152 5 1104158 3 1104166 5 1104168 4 1104175 6 1104307 4 1104309 5 1104310 6 1104311 5 1104349 7

TABLE 58 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1103202 5 1103218 7 1103253 7 1103255 7 1103256 7 1103257 6 1103258 7 1103259 6 1103268 1 1103279 6 1103285 7 1103411 7 1103471 6 1103570 6 1103722 1 1103872 4 1103933 7 1104038 7 1104116 1 1104127 3 1104144 3 1104159 5 1104276 1

Example 5: Design of MOE Gapmer Modified Oligonucleotides with Mixed PO/PS Internucleoside Linkages Complementary to a Human GFAP Nucleic Acid

Modified oligonucleotides complementary to human GFAP nucleic acid were designed. The modified oligonucleotides in the table below are 6-10-4 MOE gapmers. The gapmers are 20 nucleosides in length and have a central gap segment that consists of ten 2′-β-D-deoxynucleosides, a 5′ wing segment that consists of six 2′-β-D-MOE nucleosides, and a 3′ wing segment that consists of four 2′-β-D-MOE nucleosides. The sugar motif of the gapmers is (from 5′ to 3′): eeeeeeddddddddddeeee; wherein ‘d’ represents a 2′-β-D-deoxyribosyl sugar moiety, and ‘e’ represents a 2′-β-D-MOE sugar moiety. The gapmers have an internucleoside linkage motif of (from 5′ to 3′): sooooossssssssssoss; wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. Each cytosine nucleoside is a 5-methyl cytosine.

TABLE 59 6-10-4 MOE gapmers with mixed PO/PS internucleoside linkages complementary to human GFAP SEQ ID SEQ ID SEQ ID SEQ ID No: 1 No: 1 No: 2 No: 2 Compound Start Stop Start Stop SEQ ID No. SEQUENCE (5′ to 3′) Site Site Site Site No. 1166991 CAGCCTGGTTAGCCTTTCTG N/A N/A 9514 9533 2017 1166992 TCAGCCTGGTTAGCCTTTCT N/A N/A 9515 9534 2094 1166993 GTCAGCCTGGTTAGCCTTTC N/A N/A 9516 9535 2171 1166994 AGTCAGCCTGGTTAGCCTTT N/A N/A 9517 9536 1484 1166996 CTAGTCAGCCTGGTTAGCCT N/A N/A 9519 9538 1561 1166997 ACTAGTCAGCCTGGTTAGCC N/A N/A 9520 9539 2325 1166998 CAGTATTACCTCTACTAGTC N/A N/A 9533 9552 20 1166999 GCAGTATTACCTCTACTAGT N/A N/A 9534 9553 88 1167000 GGCAGTATTACCTCTACTAG N/A N/A 9535 9554 1637 1167001 TGGCAGTATTACCTCTACTA N/A N/A 9536 9555 166 1167002 TTGGCAGTATTACCTCTACT N/A N/A 9537 9556 1865 1167003 TTTGGCAGTATTACCTCTAC N/A N/A 9538 9557 1941 1167004 ATTTGGCAGTATTACCTCTA N/A N/A 9539 9558 2018 1167024 GTGTATTAGGATCCCATCTA N/A N/A 11155 11174 2028 1167025 TGTGTATTAGGATCCCATCT N/A N/A 11156 11175 2105 1167026 GTGTGTATTAGGATCCCATC N/A N/A 11157 11176 1569 1167027 AGTGTGTATTAGGATCCCAT N/A N/A 11158 11177 2182 1167050 CTTTATTTTTCCTCAGCGAC 3045 3064 13334 13353 361 1167051 TCTTTATTTTTCCTCAGCGA 3046 3065 13335 13354 439 1167053 TGTCTTTATTTTTCCTCAGC 3048 3067 13337 13356 595 1167054 TTGTCTTTATTTTTCCTCAG 3049 3068 13338 13357 673 1167055 TTTGTCTTTATTTTTCCTCA 3050 3069 13339 13358 751 1167056 ATTTGTCTTTATTTTTCCTC 3051 3070 13340 13359 829 1166975 TTGTGATTTTCCCCGTCTTT N/A N/A 8755 8774 908 1166984 ACATTCACTAATATTTAACA N/A N/A 9323 9342 1022 1166985 CACATTCACTAATATTTAAC N/A N/A 9324 9343 21 1166986 TCACATTCACTAATATTTAA N/A N/A 9325 9344 2321 1166987 GTCACATTCACTAATATTTA N/A N/A 9326 9345 1177 1166988 CGTCACATTCACTAATATTT N/A N/A 9327 9346 2398 1166989 ACGTCACATTCACTAATATT N/A N/A 9328 9347 2808 1166990 CACGTCACATTCACTAATAT N/A N/A 9329 9348 2809 1167057 CATTTGTCTTTATTTTTCCT 3052 3071 13341 13360 907 1167059 AGCATTTGTCTTTATTTTTC 3054 3073 13343 13362 1063 1167060 CAGCATTTGTCTTTATTTTT 3055 3074 13344 13363 1140 1166982 GCTTTTGAGATATCTTGTGA N/A N/A 8777 8796 51 1167007 TGACTATCTAGGATTTGGCA N/A N/A 9551 9570 2480 1167011 CCTGTGACTATCTAGGATTT N/A N/A 9555 9574 2709 1167012 TTTATCTGTGCTTTAGTGAC N/A N/A 9574 9593 2710 1167017 TGCCATTTATCTGTGCTTTA N/A N/A 9579 9598 1639 1167018 CTGCCATTTATCTGTGCTTT N/A N/A 9580 9599 1867 1174062 GCTTTTGCCCCCTGTAGTGA N/A N/A 8732 8751 2291 1174063 GTGCTTTTGCCCCCTGTAGT N/A N/A 8734 8753 2368 1174065 TGGTGCTTTTGCCCCCTGTA N/A N/A 8736 8755 2445 1174066 TTGGTGCTTTTGCCCCCTGT N/A N/A 8737 8756 59 1167033 GCACAGTTCCCAGATACTCC 1756 1775 12045 12064 426 1167034 GGCACAGTTCCCAGATACTC 1757 1776 12046 12065 504 1167035 AGGCACAGTTCCCAGATACT 1758 1777 12047 12066 1425 1167036 AAGGCACAGTTCCCAGATAC 1759 1778 12048 12067 1502 1167037 AAAGGCACAGTTCCCAGATA 1760 1779 12049 12068 582 1167040 CTCAGTTTTCCTCCAGCAGC 1792 1811 12081 12100 115 1167046 GCCAGTGTCTTCACTTTGCT 2750 2769 13039 13058 1973 1167058 GCATTTGTCTTTATTTTTCC 3053 3072 13342 13361 985

Modified oligonucleotides in the table below are 5-10-5 MOE gapmers with mixed PO/PS internucleoside linkages. The gapmers are 20 nucleosides in length, wherein the central gap segment consists of ten 2′-β-D-deoxynucleosides and the 5′ and 3′ wing segments each consist of five 2′-β-D-MOE modified nucleosides. The sugar motif for the gapmers is (from 5′ to 3′): eeeeeddddddddddeeeee; wherein represents a 2′-β-D-deoxyribosyl sugar moiety, and ‘e’ represents a 2′-β-D-MOE sugar moiety. The gapmers have an internucleoside linkage motif of (from 5′ to 3′): soooossssssssssooss; wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. Each cytosine nucleoside is a 5-methyl cytosine.

TABLE 60 5-10-5 MOE gapmers with a mixed PO/PS internucleoside linkages complementary to human GFAP SEQ ID SEQ ID SEQ ID SEQ ID No: 1 No: 1 No: 2 No: 2 Compound Start Stop Start Stop SEQ ID No. SEQUENCE (5′ to 3′) Site Site Site Site No. 1166900 CAGCCTGGTTAGCCTTTCTG N/A N/A 9514 9533 2017 1166901 TCAGCCTGGTTAGCCTTTCT N/A N/A 9515 9534 2094 1166902 GTCAGCCTGGTTAGCCTTTC N/A N/A 9516 9535 2171 1166903 AGTCAGCCTGGTTAGCCTTT N/A N/A 9517 9536 1484 1166904 TAGTCAGCCTGGTTAGCCTT N/A N/A 9518 9537 2248 1166905 CTAGTCAGCCTGGTTAGCCT N/A N/A 9519 9538 1561 1166906 ACTAGTCAGCCTGGTTAGCC N/A N/A 9520 9539 2325 1166907 CAGTATTACCTCTACTAGTC N/A N/A 9533 9552 20 1166909 GGCAGTATTACCTCTACTAG N/A N/A 9535 9554 1637 1166910 TGGCAGTATTACCTCTACTA N/A N/A 9536 9555 166 1166911 TTGGCAGTATTACCTCTACT N/A N/A 9537 9556 1865 1166912 TTTGGCAGTATTACCTCTAC N/A N/A 9538 9557 1941 1166913 ATTTGGCAGTATTACCTCTA N/A N/A 9539 9558 2018 1166932 TGTATTAGGATCCCATCTAG N/A N/A 11154 11173 1951 1166933 GTGTATTAGGATCCCATCTA N/A N/A 11155 11174 2028 1166934 TGTGTATTAGGATCCCATCT N/A N/A 11156 11175 2105 1166885 CTTGTGATTTTCCCCGTCTT N/A N/A 8756 8775 986 1166886 CCTTGTGATTTTCCCCGTCT N/A N/A 8757 8776 1064 1166887 TTGAGATATCTTGTGACCTT N/A N/A 8773 8792 1510 1166888 TTTGAGATATCTTGTGACCT N/A N/A 8774 8793 1280 1166890 CTTTTGAGATATCTTGTGAC N/A N/A 8776 8795 1434 1166893 ACATTCACTAATATTTAACA N/A N/A 9323 9342 1022 1166894 CACATTCACTAATATTTAAC N/A N/A 9324 9343 21 1166895 TCACATTCACTAATATTTAA N/A N/A 9325 9344 2321 1166896 GTCACATTCACTAATATTTA N/A N/A 9326 9345 1177 1166897 CGTCACATTCACTAATATTT N/A N/A 9327 9346 2398 1166898 ACGTCACATTCACTAATATT N/A N/A 9328 9347 2808 1166899 CACGTCACATTCACTAATAT N/A N/A 9329 9348 2809 1166916 TGACTATCTAGGATTTGGCA N/A N/A 9551 9570 2480 1166920 CCTGTGACTATCTAGGATTT N/A N/A 9555 9574 2709 1166926 TGCCATTTATCTGTGCTTTA N/A N/A 9579 9598 1639 1166927 CTGCCATTTATCTGTGCTTT N/A N/A 9580 9599 1867 1166928 TCTGCCATTTATCTGTGCTT N/A N/A 9581 9600 400 1166929 CTCTGCCATTTATCTGTGCT N/A N/A 9582 9601 1943 1174056 GCTTTTGCCCCCTGTAGTGA N/A N/A 8732 8751 2291 1174058 GGTGCTTTTGCCCCCTGTAG N/A N/A 8735 8754 1227 1174059 TGGTGCTTTTGCCCCCTGTA N/A N/A 8736 8755 2445 1174060 TTGGTGCTTTTGCCCCCTGT N/A N/A 8737 8756 59 1174061 TTTGGTGCTTTTGCCCCCTG N/A N/A 8738 8757 2522 1166940 ACAGTTCCCAGATACTCCGA 1754 1773 12043 12062 270 1166946 AAAGGCACAGTTCCCAGATA 1760 1779 12049 12068 582 1166948 TCAGTTTTCCTCCAGCAGCC 1791 1810 12080 12099 37 1166954 CCAGTGTCTTCACTTTGCTC 2749 2768 13038 13057 825 1199982 GGTCCTAAATATTCTAGTCC N/A N/A 9459 9478 2093 1199983 TGGTCCTAAATATTCTAGTC N/A N/A 9460 9479 2170 1199984 GTGGTCCTAAATATTCTAGT N/A N/A 9461 9480 2813

The modified oligonucleotides in the table below are 4-10-6 MOE gapmers. The gapmers are 20 nucleosides in length and have a central gap segment that consists of ten 2′-β-D-deoxynucleosides, a 5′ wing segment that consists of four 2′-β-D-MOE nucleosides, and a 3′ wing segment that consists of six 2′-β-D-MOE nucleosides. The sugar motif of the gapmers is (from 5′ to 3′): eeeeddddddddddeeeeee; wherein ‘d’ represents a 2′-β-D-deoxyribosyl sugar moiety, and ‘e’ represents a 2′-β-D-MOE sugar moiety. The gapmers have an internucleoside linkage motif of (from 5′ to 3′): sooossssssssssoooss; wherein “s” represents a phosphorothioate internucleoside linkage and “o” represents a phosphodiester internucleoside linkage. Each cytosine nucleoside is a 5-methyl cytosine.

TABLE 61 4-10-6 MOE gapmers with a mixed PO/PS internucleoside linkages complementary to human GFAP SEQ ID SEQ ID SEQ ID SEQ ID No: 1 No: 1 No: 2 No: 2 Compound Start Stop Start Stop SEQ ID No. SEQUENCE (5′ to 3′) Site Site Site Site No. 1166809 CAGCCTGGTTAGCCTTTCTG N/A N/A 9514 9533 2017 1166810 TCAGCCTGGTTAGCCTTTCT N/A N/A 9515 9534 2094 1166812 AGTCAGCCTGGTTAGCCTTT N/A N/A 9517 9536 1484 1166813 TAGTCAGCCTGGTTAGCCTT N/A N/A 9518 9537 2248 1166814 CTAGTCAGCCTGGTTAGCCT N/A N/A 9519 9538 1561 1166815 ACTAGTCAGCCTGGTTAGCC N/A N/A 9520 9539 2325 1166816 CAGTATTACCTCTACTAGTC N/A N/A 9533 9552 20 1166817 GCAGTATTACCTCTACTAGT N/A N/A 9534 9553 88 1166818 GGCAGTATTACCTCTACTAG N/A N/A 9535 9554 1637 1166819 TGGCAGTATTACCTCTACTA N/A N/A 9536 9555 166 1166820 TTGGCAGTATTACCTCTACT N/A N/A 9537 9556 1865 1166821 TTTGGCAGTATTACCTCTAC N/A N/A 9538 9557 1941 1166822 ATTTGGCAGTATTACCTCTA N/A N/A 9539 9558 2018 1166841 TGTATTAGGATCCCATCTAG N/A N/A 11154 11173 1951 1166842 GTGTATTAGGATCCCATCTA N/A N/A 11155 11174 2028 1166843 TGTGTATTAGGATCCCATCT N/A N/A 11156 11175 2105 1166845 AGTGTGTATTAGGATCCCAT N/A N/A 11158 11177 2182 1166846 GAGTGTGTATTAGGATCCCA N/A N/A 11159 11178 2259 1166847 AGAGTGTGTATTAGGATCCC N/A N/A 11160 11179 2336 1166869 TCTTTATTTTTCCTCAGCGA 3046 3065 13335 13354 439 1166870 GTCTTTATTTTTCCTCAGCG 3047 3066 13336 13355 517 1166871 TGTCTTTATTTTTCCTCAGC 3048 3067 13337 13356 595 1166872 TTGTCTTTATTTTTCCTCAG 3049 3068 13338 13357 673 1166873 TTTGTCTTTATTTTTCCTCA 3050 3069 13339 13358 751 1166874 ATTTGTCTTTATTTTTCCTC 3051 3070 13340 13359 829 1166793 TTGTGATTTTCCCCGTCTTT N/A N/A 8755 8774 908 1166794 CTTGTGATTTTCCCCGTCTT N/A N/A 8756 8775 986 1166795 CCTTGTGATTTTCCCCGTCT N/A N/A 8757 8776 1064 1166798 TTTTGAGATATCTTGTGACC N/A N/A 8775 8794 1357 1166799 CTTTTGAGATATCTTGTGAC N/A N/A 8776 8795 1434 1166800 GCTTTTGAGATATCTTGTGA N/A N/A 8777 8796 51 1166802 ACATTCACTAATATTTAACA N/A N/A 9323 9342 1022 1166803 CACATTCACTAATATTTAAC N/A N/A 9324 9343 21 1166804 TCACATTCACTAATATTTAA N/A N/A 9325 9344 2321 1166805 GTCACATTCACTAATATTTA N/A N/A 9326 9345 1177 1166806 CGTCACATTCACTAATATTT N/A N/A 9327 9346 2398 1166807 ACGTCACATTCACTAATATT N/A N/A 9328 9347 2808 1166808 CACGTCACATTCACTAATAT N/A N/A 9329 9348 2809 1166867 TTTATTTTTCCTCAGCGACT 3044 3063 13333 13352 283 1166875 CATTTGTCTTTATTTTTCCT 3052 3071 13341 13360 907 1166877 AGCATTTGTCTTTATTTTTC 3054 3073 13343 13362 1063 1166878 CAGCATTTGTCTTTATTTTT 3055 3074 13344 13363 1140 1166823 ACTATCTAGGATTTGGCAGT N/A N/A 9549 9568 2326 1166826 GTGACTATCTAGGATTTGGC N/A N/A 9552 9571 1408 1166831 ATTTATCTGTGCTTTAGTGA N/A N/A 9575 9594 2786 1166835 TGCCATTTATCTGTGCTTTA N/A N/A 9579 9598 1639 1174050 GCTTTTGCCCCCTGTAGTGA N/A N/A 8732 8751 2291 1174051 GTGCTTTTGCCCCCTGTAGT N/A N/A 8734 8753 2368 1174053 TGGTGCTTTTGCCCCCTGTA N/A N/A 8736 8755 2445 1174054 TTGGTGCTTTTGCCCCCTGT N/A N/A 8737 8756 59 1166852 GGCACAGTTCCCAGATACTC 1757 1776 12046 12065 504

The modified oligonucleotides in the table below are 5-8-5 MOE gapmers. The gapmers are 20 nucleosides in length and have a central gap segment that consists of eight 2′-β-D-deoxynucleosides, a 5′ wing segment that consists of five 2′-β-D-MOE nucleosides, and a 3′ wing segment that consists of five 2′-β-D-MOE nucleosides. The sugar motif of the gapmers is (from 5′ to 3′): eeeeeddddddddeeeee; wherein ‘d’ represents a 2′-β-D-deoxyribosyl sugar moiety, and ‘e’ represents a 2′-β-D-MOE sugar moiety. The gapmers have an internucleoside linkage motif of (from 5′ to 3′): sooosssssssssooss; wherein each “s” represents a phosphorothioate internucleoside linkage and each “o” represents a phosphodiester internucleoside linkage. Each cytosine nucleobase is a 5-methylcytosine.

TABLE 62 5-8-5 MOE gapmers with a mixed PO/PS internucleoside linkages complementary to human GFAP SEQ ID SEQ ID SEQ ID SEQ ID No: 1 No: 1 No: 2 No: 2 Compound Start Stop Start Stop SEQ ID No. SEQUENCE (5′ to 3′) Site Site Site Site No. 1166719 GCAGTATTACCTCTACTA N/A N/A 9536 9553 2816 1166720 GGCAGTATTACCTCTACT N/A N/A 9537 9554 2817 1166721 TGGCAGTATTACCTCTAC N/A N/A 9538 9555 2818 1166749 TCAGCCTGGTTAGCCTTT N/A N/A 9517 9534 2819 1166750 GTCAGCCTGGTTAGCCTT N/A N/A 9518 9535 2820 1166751 AGTCAGCCTGGTTAGCCT N/A N/A 9519 9536 2821 1166761 GTGTATTAGGATCCCATC N/A N/A 11157 11174 2822 1166762 TGTGTATTAGGATCCCAT N/A N/A 11158 11175 2823 1166763 GTGTGTATTAGGATCCCA N/A N/A 11159 11176 2824 1166775 TTATTTTTCCTCAGCGAC 3045 3062 13334 13351 2825 1166776 TTTATTTTTCCTCAGCGA 3046 3063 13335 13352 2826 1166777 CTTTATTTTTCCTCAGCG 3047 3064 13336 13353 2827 1166778 TCTTTATTTTTCCTCAGC 3048 3065 13337 13354 2828 1166779 GTCTTTATTTTTCCTCAG 3049 3066 13338 13355 2829 1166780 TGTCTTTATTTTTCCTCA 3050 3067 13339 13356 2830 1166781 TTGTCTTTATTTTTCCTC 3051 3068 13340 13357 2831 1166782 TTTGTCTTTATTTTTCCT 3052 3069 13341 13358 2832 1166783 ATTTGTCTTTATTTTTCC 3053 3070 13342 13359 2833 1166738 GTGATTTTCCCCGTCTTT N/A N/A 8755 8772 2835 1166739 TGTGATTTTCCCCGTCTT N/A N/A 8756 8773 2836 1166740 GAGATATCTTGTGACCTT N/A N/A 8773 8790 2837 1166742 TTGAGATATCTTGTGACC N/A N/A 8775 8792 2839 1166746 CACATTCACTAATATTTA N/A N/A 9326 9343 2840 1166747 TCACATTCACTAATATTT N/A N/A 9327 9344 2841 1166748 GTCACATTCACTAATATT N/A N/A 9328 9345 2842 1166784 CATTTGTCTTTATTTTTC 3054 3071 13343 13360 2843 1166785 GCATTTGTCTTTATTTTT 3055 3072 13344 13361 2844 1166786 AGCATTTGTCTTTATTTT 3056 3073 13345 13362 2845 1166787 CAGCATTTGTCTTTATTT 3057 3074 13346 13363 2846 1174012 TTGTGATTTTCCCCGTCT N/A N/A 8757 8774 2850 1174013 TTTGAGATATCTTGTGAC N/A N/A 8776 8793 2851 1174015 ATTCACTAATATTTAACA N/A N/A 9323 9340 2852 1174016 CATTCACTAATATTTAAC N/A N/A 9324 9341 2853 1174018 CGTCACATTCACTAATAT N/A N/A 9329 9346 2854 1174020 AGCCTGGTTAGCCTTTCT N/A N/A 9515 9532 2856 1174024 AGTATTACCTCTACTAGT N/A N/A 9534 9551 2859 1174026 TTGGCAGTATTACCTCTA N/A N/A 9539 9556 2861 1174036 TATTAGGATCCCATCTAG N/A N/A 11154 11171 2862 1174037 GTATTAGGATCCCATCTA N/A N/A 11155 11172 2863 1166744 GCTTTTGAGATATCTTGT N/A N/A 8779 8796 2866 1166757 CCATTTATCTGTGCTTTA N/A N/A 9579 9596 2873 1166758 TGCCATTTATCTGTGCTT N/A N/A 9581 9598 2874 1166759 CTGCCATTTATCTGTGCT N/A N/A 9582 9599 2875 1166760 TCTGCCATTTATCTGTGC N/A N/A 9583 9600 2876 1174029 ACTATCTAGGATTTGGCA N/A N/A 9551 9568 2886 1174030 TGTGACTATCTAGGATTT N/A N/A 9555 9572 2887 1174031 TATCTGTGCTTTAGTGAC N/A N/A 9574 9591 2888 1174034 GCCATTTATCTGTGCTTT N/A N/A 9580 9597 2891

Example 6: Tolerability of Modified Oligonucleotides Complementary to Human GFAP in Wild-Type Mice, 3 Hour Study

Modified oligonucleotides described above were tested in wild-type female C57/B16 mice to assess the tolerability of the oligonucleotides. Wild-type female C57/B16 mice each received a single ICV dose of 700 μg of modified oligonucleotide listed in the table below. Each treatment group consisted of 4 mice. A group of 4 mice received PBS as a negative control for each experiment (identified in separate tables below). At 3 hours post-injection, mice were evaluated according to seven different criteria. The criteria are (1) the mouse was bright, alert, and responsive; (2) the mouse was standing or hunched without stimuli; (3) the mouse showed any movement without stimuli; (4) the mouse demonstrated forward movement after it was lifted; (5) the mouse demonstrated any movement after it was lifted; (6) the mouse responded to tail pinching; (7) regular breathing. For each of the 7 criteria, a mouse was given a subscore of 0 if it met the criteria and 1 if it did not (the functional observational battery score or FOB). After all 7 criteria were evaluated, the scores were summed for each mouse and averaged within each treatment group. The results are presented in the tables below.

TABLE 63 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166719 1 1166720 0 1166721 0 1166749 3 1166750 1 1166751 2 1166761 4 1166762 4 1166763 3 1166775 0 1166776 1 1166777 4 1166778 1 1166779 4 1166780 1 1166781 0 1166782 0 1166783 0 1166809 4 1166810 4 1166812 3 1166813 3

TABLE 64 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166814 4 1166815 2 1166816 1 1166817 1 1166818 3 1166819 1 1166820 1 1166821 1 1166822 3 1166841 3 1166842 3 1166843 3 1166845 5 1166846 4 1166847 5 1166869 1 1166870 3 1166871 2 1166872 2 1166873 1 1166874 0 1166900 5

TABLE 65 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166901 4 1166902 5 1166903 4 1166904 4 1166905 2 1166906 2 1166907 0 1166909 3 1166910 1 1166911 0 1166912 1 1166913 2 1166932 4 1166933 2 1166934 3 1166991 4 1166992 4 1166993 4 1166994 3

TABLE 66 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166996 4 1166997 4 1166998 0 1166999 0 1167000 3 1167001 0 1167002 0 1167003 0 1167004 0 1167024 4 1167025 3 1167026 4 1167027 4 1167050 1 1167051 0 1167053 1 1167054 4 1167055 0 1167056 0

TABLE 67 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166738 0 1166739 0 1166740 4 1166742 3 1166746 0 1166747 0 1166748 0 1166784 0 1166785 1 1166786 3 1166787 3 1166793 0 1166794 0 1166795 1 1166797 4 1166798 3

TABLE 68 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166799 1 1166802 0 1166803 0 1166804 0 1166805 0 1166806 0 1166807 0 1166808 0 1166867 0 1166877 1 1166878 2 1166885 3 1166886 0 1166887 3 1166888 3 1166890 0 1166893 0

TABLE 69 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166894 0 1166895 0 1166896 0 1166897 0 1166898 0 1166899 0 1166984 0 1166985 0 1166986 0 1166987 0 1166988 0 1166990 0

TABLE 70 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1167057 0 1167059 1 1167060 1 1174012 2 1174013 3 1174015 0 1174016 0 1174018 0 1174020 4 1174024 3 1174026 0 1174036 3 1174037 2

TABLE 71 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166744 3 1166757 4 1166758 3 1166759 1 1166760 3 1166800 1 1166823 2 1166826 3 1166831 2 1166875 0 1166975 4 1166989 0

TABLE 72 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166835 0 1166916 3 1166920 2 1166926 1 1166927 2 1166928 1 1166929 0 1166982 2

TABLE 73 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1167007 2 1167011 1 1167012 3 1167017 2 1167018 1 1174029 4

TABLE 74 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1174030 3 1174031 3 1174034 1 1174050 3 1174051 3 1174053 1 1174054 1 1174056 4 1174058 3 1174059 0 1174060 0 1174061 2 1174062 3 1174063 3 1174065 1 1174066 1

TABLE 75 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166852 1

TABLE 76 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166940 2 1166946 2 1166948 2

TABLE 77 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1166954 0 1167033 0 1167034 1 1167035 1 1167036 2 1167037 2 1167040 2

TABLE 78 Tolerability scores in mice at 700 μg dose Compound No. 3 hr. FOB PBS 0 1167046 1 1167058 1

TABLE 79 Tolerability scores in mice at 700 μg dose 3 hr. Compound No. FOB PBS 0 1199982 1 1199983 1 1199984 1

Example 7: Activity of Modified Oligonucleotides Complementary to Human GFAP in Transgenic Mice

Modified oligonucleotides described above were tested in human GFAP transgenic mouse model. Transgenic mice (line 73.7) was previously described in Messing A., et al., Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice, Am J Pathos. 1998, 152(2):391-398.

Treatment

The GFAP transgenic mice were divided into groups of 3-4 mice each. Each mouse received a single ICV bolus of 300 μg of modified oligonucleotide. A group of 3-4 mice received PBS as a negative control.

RNA Analysis

One week post treatment, mice were sacrificed and RNA was extracted from cortical brain tissue, and/or spinal cord for RTPCR analysis to measure amount of GFAP RNA using primer probe set RTS38621 ((forward sequence AGTTGCAGTCCTTGACCTG, designated herein as SEQ ID NO: 14; reverse sequence CAGCGCCTCCTGATAACTG, designated herein as SEQ ID NO: 15; probe sequence ACGAGTCCCTGGAGAGGCAGATG, designated herein as SEQ ID NO: 16), which is a human specific primer-probe set that recognizes all isoforms of GFAP. Results are presented as percent human GFAP RNA relative to PBS control, normalized to mouse peptidylprolyl isomerase A (PPIA), also known as cyclophilin A. Mouse PPIA was amplified using primer probe set m_cyclo24 (forward sequence TCGCCGCTTGCTGCA, designated herein as SEQ ID NO: 17; reverse sequence ATCGGCCGTGATGTCGA, designated herein as SEQ ID NO: 18; probe sequence CCATGGTCAACCCCACCGTGTTC, designated herein as SEQ ID NO: 19). In some cases, RTPCR value is not defined for a certain sample, and is labeled N.D. (Not Defined).

As shown in the table below, treatment with modified oligonucleotides resulted in reduction of GFAP RNA in comparison to the PBS control.

TABLE 80 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. SPINAL CORD CORTEX PBS 100 100 1047198 44 44 1047362 15 21 1047373 10 16 1047374 8 16 1047386 14 19 1047394 48 43 1047429 N.D. N.D. 1047448 37 45 1047500 23 26 1047580 12 15 1047582 5 8 1047583 6 9 1047584 6 7 1047585 8 7 1047586 12 11 1047587 13 13 1047589 9 13 1047590 16 16 1047591 14 17 1047610 33 31 1048203 26 27 1048350 28 24

TABLE 81 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. SPINAL CORD CORTEX PBS 100  100  1047211 71 66 1047225 65 61 1047298 34 27 1047316 29 24 1047402 30 25 1047432 28 29 1047518 49 34 1047522 23 19 1047532 40 30 1047579 27 17 1047581 11 10 1047598 77 50 1047599 21 15 1047601 47 30 1047602 N.D. N.D. 1047609 27 18 1047662 34 27 1047679 58 54

TABLE 82 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. SPINAL CORD CORTEX PBS 100 100 1047711 45 38 1047884 57 62 1047913 75 66 1048027 64 53 1048151 69 64 1048182 14 10 1048201 6 7 1048204 21 18 1048227 42 28 1072855 N.D. N.D. 1072856 N.D. N.D. 1072857 28 14 1072861 28 23 1072862 53 33 1072863 51 28 1072886 42 26 1072980 40 25 1072986 98 88 1073033 100 80 1073045 36 24 1073060 19 16 1073063 19 15 1073064 29 20 1073065 16 15 1073093 11 8

TABLE 83 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. SPINAL CORD CORTEX PBS 100 100 1103217 56 43 1103465 31 19 1103502 47 31 1103516 42 27 1103567 23 18 1104093 13 12 1104118 N.D. N.D. 1104145 8 6 1104152 27 17 1104166 17 13 1104175 15 8

TABLE 84 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. SPINAL CORD CORTEX PBS 100  100  1072813 15 18 1072814 15 19 1103202 34 36 1103218 48 33 1103253 56 36 1103255 N.D. N.D. 1103256 35 41 1103257 46 43 1103258 N.D. N.D. 1103259 82 71 1103268 N.D. N.D. 1104307 13 14 1104309 10 11 1104311 11 18 1104349 N.D. N.D.

TABLE 85 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. SPINAL CORD CORTEX PBS 100 100 1103279 20 14 1103285 16 9 1103411 65 32 1103471 32 39 1103570 6 4 1103872 42 18 1104116 6 3 1104127 15 10 1104144 17 8 1104159 17 10 1104276 53 41 1166719 41 31 1166720 12 8 1166721 7 8 1166749 13 11 1166750 10 7 1166751 12 9 1166761 12 5

TABLE 86 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1166762 18 1166763 13 1166775 21 1166776 30 1166777 13 1166778 6 1166779 7 1166780 9 1166781 7 1166782 12 1166783 17 1166809 13 1166810 16 1166812 23 1166813 21 1166814 29 1166815 42 1166816 31 1166817 37 1166818 14 1166819 9 1166820 12 1166821 15 1166822 20 1166841 47

TABLE 87 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. SPINAL CORD CORTEX PBS 100 100 1166842 19 14 1166843 37 21 1166845 17 19 1166846 12 18 1166847 13 19 1166869 10 11 1166870 6 7 1166871 5 7 1166872 10 9 1166873 14 14 1166874 9 10 1166900 14 20 1166901 11 13 1166902 12 15 1166903 17 19 1166904 26 30 1166905 24 28 1166906 50 56 1166907 28 32

TABLE 88 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1166909 30 1166910 8 1166911 7 1166912 15 1166913 12 1166932 41 1166933 13 1166934 18 1166991 14 1166992 11 1166993 9 1166994 10 1166996 21 1166997 30 1166998 13 1166999 11 1167000 15

TABLE 89 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1167001 9 1167002 7 1167003 8 1167004 13 1167024 18 1167025 11 1167026 14 1167027 13 1167050 11 1167051 10 1167053 7 1167054 5 1167055 6 1167056 6

TABLE 90 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1166738 27 1166739 27 1166740 26 1166742 43 1166746 94 1166747 23 1166748 20 1166784 30 1166785 13 1166786 19 1166787 19 1166793 23 1166794 32 1166795 51 1166798 25 1166799 33 1166802 38 1166803 31 1166804 29 1166805 7 1166806 23 1166807 19 1166808 14 1166867 13 1166877 7 1166878 13 1166885 20 1166886 25

TABLE 91 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1166887 12 1166888 16 1166890 38 1166893 39 1166894 20 1166895 11 1166896 8 1166897 6 1166898 8 1166899 11 1166984 24 1166985 9 1166986 13 1166987 7 1166988 5 1166990 9 1167057 7 1167059 5 1167060 8 1174012 9 1174013 33 1174015 107

TABLE 92 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1166744 14 1166757 7 1166758 34 1166759 9 1166760 16 1166800 8 1166823 7 1166826 6 1166831 16 1166835 47 1166875 7 1166916 14 1166920 18 1166975 6 1166989 4 1174016 66 1174018 11 1174020 12 1174024 52 1174026 7 1174036 43 1174037 24

TABLE 93 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1166926 14 1166927 15 1166928 13 1166929 15 1166982 11 1167007 44 1167011 21 1167012 23 1167017 8 1167018 11 1174029 32 1174030 75 1174031 35 1174034 17 1174050 10 1174051 15 1174053 22 1174054 40 1174056 7 1174058 14 1174059 14 1174060 19 1174061 17 1174062 12 1174063 12

TABLE 94 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1166852 15 1166940 16 1166946 34 1166948 41 1166954 16 1167033 9 1167034 11 1167035 15 1167036 19 1167037 25 1167040 15

TABLE 95 Reduction of human GFAP RNA in transgenic mice GFAP RNA (% control) Compound No. CORTEX PBS 100 1167046 14 1167058 5 1174065 10 1174066 13 1199982 4 1199983 13 1199984 16

Example 8: Tolerability of Modified Oligonucleotides Complementary to Human GFAP in Rats, 3 mg Dose

Modified oligonucleotides described above were tested in rats to assess the tolerability of the oligonucleotides. Sprague Dawley rats each received a single intrathecal (IT) dose of 3 mg of oligonucleotide listed in the table below. Each treatment group consisted of 2-4 rats. A group of 2-4 rats received PBS as a negative control. At 3 hours post-injection, movement in 7 different parts of the body were evaluated for each rat. The 7 body parts are (1) the rat's tail; (2) the rat's posterior posture; (3) the rat's hind limbs; (4) the rat's hind paws; (5) the rat's forepaws; (6) the rat's anterior posture; (7) the rat's head. For each of the 7 different body parts, each rat was given a sub-score of 0 if the body part was moving or 1 if the body part was paralyzed (the functional observational battery score or FOB). After each of the 7 body parts were evaluated, the sub-scores were summed for each rat and then averaged for each group. For example, if a rat's tail, head, and all other evaluated body parts were moving 3 hours after the 3 mg IT dose, it would get a summed score of 0. If another rat was not moving its tail 3 hours after the 3 mg IT dose but all other evaluated body parts were moving, it would receive a score of 1. Results are presented as the average score for each treatment group.

TABLE 96 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1047198 4 1047362 3 1047386 5 1047580 2 1047583 1 1047584 2 1047585 0 1047586 0 1047587 0 1047589 2 1047590 2 1047591 4 1048350 3

TABLE 97 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1047429 2 1047448 1 1047500 3 1047581 2 1047599 2 1047609 5 1047913 4 1048027 3 1048151 2

TABLE 98 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1047316 4 1047402 3 1047522 0 1047532 3 1047579 4 1048201 2 1048204 0

TABLE 99 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1047298 3 1072813 0 1072814 4 1072855 4 1072857 4 1104116 0 1104145 2 1104175 4 1166721 0 1166750 4 1166751 4 1166871 2

TABLE 100 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1166720 1 1166775 2 1166778 2 1166780 2 1166781 1 1166782 1 1166783 1 1166819 2 1166820 3 1166821 3 1166842 4 1166869 2 1166872 2 1166873 0 1166874 0

TABLE 101 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1166748 0 1166785 2 1166793 1 1166805 1 1166806 2 1166807 0 1166910 3 1166911 2 1166912 3 1166933 3 1166998 2 1167050 4 1167053 3 1167055 2 1167056 1

TABLE 102 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1166808 0 1166867 1 1166877 2 1166878 3 1166894 1 1166895 0 1166896 2 1166897 2 1166898 0 1166899 0 1166985 0 1166990 0

TABLE 103 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1166759 2 1166760 2 1166800 3 1166823 4 1166831 5 1166875 1 1166920 3 1166926 2 1166927 2 1166928 2 1166982 3 1166986 1 1166987 1 1166988 5 1166989 1 1167001 3

TABLE 104 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1167002 2 1167003 3 1167017 3 1167018 3 1167057 1 1167059 2 1167060 2 1174012 3 1174018 1 1174026 2 1174034 3 1174059 1 1174060 1 1174061 4

TABLE 105 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1166852 3 1166940 2 1166946 2 1166948 3 1166954 2

TABLE 106 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1167033 4 1167034 3 1167035 3 1167036 2 1167037 2 1167040 3 1167046 3 1167058 2

TABLE 107 Tolerability scores in rats at 3 mg dose FOB Compound No. 3 hr PBS 0 1174065 1 1174066 2 1199982 1 1199983 1 1199984 2

Example 9: Potency of Modified Oligonucleotides Complementary to Human GFAP in Transgenic Mice

Modified oligonucleotides described above were tested in human GFAP transgenic mouse model. Transgenic mice (line 73.7) was previously described in Messing A., et al., Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice, Am J Pathos. 1998, 152(2):391-398.

Treatment

The GFAP transgenic mice were divided into groups of 4 mice each. Each mouse received a single ICV bolus of modified oligonucleotide at the doses indicated in tables below. A group of 4-8 mice received PBS as a negative control.

RNA Analysis

Two weeks post treatment, mice were sacrificed, and RNA was extracted from the cortex, spinal cord, and brainstem for RTPCR analysis of RNA expression of GFAP using primer probe set RTS38621, described in Example 6 above. Results are presented as percent change of RNA, relative to PBS control, normalized to mouse PPIA. The half maximal effective dose (ED₅₀) of each modified oligonucleotide was calculated using GraphPad Prism 6 software (GraphPad Software, San Diego, Calif.). ED₅₀ values were calculated from dose and individual animal GFAP mRNA levels using custom equation Motulsky: Agonist vs response—Variable slope (four parameters) Y=Bottom+(Top−Bottom)/(1+(10{circumflex over ( )} log EC50/X){circumflex over ( )}HillSlope), with the following constraints: bottom>lowest value in data set in order to compare across ASOs (3, 5, and 3 for cortex, spinal cord, and brainstem, respectively), top=100, HillSlope <−1 and >−2.

As shown in the table below, treatment with modified oligonucleotides resulted in dose-responsive reduction of GFAP RNA in comparison to the PBS control.

TABLE 108 Reduction of human GFAP RNA (all isoforms) in transgenic mice GFAP RNA (% control) ED₅₀ Compound No. Dose (μg) CORTEX (μg) PBS N/A 100 N/A 1104145 1 77 8 3 74 10 53 30 19 100 8

TABLE 109 Reduction of human GFAP RNA (all isoforms) in transgenic mice GFAP RNA (% control) ED₅₀ (μg) Compound SPINAL BRAIN SPINAL BRAIN No. Dose (μg) CORTEX CORD STEM CORTEX CORD STEM PBS N/A 100 100 100 N/A N/A N/A 1072813 3 93 101 99 34 24 21 10 68 71 69 30 70 38 37 100 18 28 21 300 10 19 13 1166721 1 116 111 94 25 25 12 3 95 104 89 10 91 73 51 30 39 47 28 100 13 18 11 300 7 10 6 1166874 1 81 102 78 27 11 7 3 73 83 75 10 77 50 45 30 65 32 22 100 12 14 10 300 5 9 6 1166895 3 108 93 93 41 93 38 10 82 94 80 30 46 74 50 100 40 40 32

TABLE 110 Reduction of human GFAP RNA (all isoforms) in transgenic mice GFAP RNA (% control) ED50 (μg) Compound SPINAL BRAIN SPINAL BRAIN No. Dose (μg) CORTEX CORD STEM CORTEX CORD STEM PBS N/A 100 100 100 N/A N/A N/A 1166895 300 16 45 27 N/A N/A N/A 3 103 77 77 1166926 10 86 70 57 47 32 17 30 67 62 49 100 27 27 20 300 13 21 11 1166985 1 105 101 100 46 36 14 3 96 90 78 10 75 68 53 30 79 58 40 100 17 35 15 300 8 23 10 1167056 1 102 87 86 10 5 3 3 81 60 42 10 54 35 20 30 20 16 9 100 5 8 5 300 3 7 4 1167058 1 98 82 80 19 4 3 3 79 59 44 10 79 21 20

TABLE 111 Reduction of human GFAP RNA (all isoforms) in transgenic mice GFAP RNA (% control) ED50 (μg) Compound SPINAL BRAIN SPINAL BRAIN No. Dose (μg) CORTEX CORD STEM CORTEX CORD STEM PBS N/A 100 100 100 N/A N/A N/A 1166927 3 91 92 89 44 24 15 10 98 62 57 30 62 52 35 100 25 26 14 300 14 20 12 1166954 3 106 87 85 58 60 26 10 79 80 68 30 70 65 49 100 36 46 27 300 20 25 17 1166986 3 114 113 101 35 42 21 10 87 88 66 30 52 56 38 100 23 30 20 300 10 20 14 1166998 3 77 73 61 23 13  6 10 90 63 42 30 36 31 20 1167058 30 34 14 9 N/A N/A N/A 100 9 7 6 300 5 6 4

TABLE 112 Reduction of human GFAP RNA (all isoforms) in transgenic mice GFAP RNA (% control) RTS38621 ED50 (μg) Compound SPINAL BRAIN SPINAL BRAIN No. Dose (μg) CORTEX CORD STEM CORTEX CORD STEM PBS 100 100 100 N/A N/A N/A 1166998 100 11 18 17 N/A N/A N/A 300 7 12 9 N/A N/A N/A 1199983 3 75 83 71 18 16 15 10 72 57 62 30 37 38 38 100 17 27 22 300 12 22 17

Example 10: Effect of Modified Oligonucleotides on Human GFAP RNA In Vitro, Multiple Doses

Modified oligonucleotides selected from the examples above were tested at various doses in U251 cells. Cultured U251 cells at a density of 30,000 cells per well were treated with electroporation with various concentrations of modified oligonucleotide as specified in the tables below. After a treatment period of approximately 24 hours, total RNA was isolated from the cells and GFAP RNA levels were measured by quantitative real-time RTPCR. Human GFAP primer probe sets RTS38621 (described herein above), RTS38624 (forward sequence AACCGGATCACCATTCCC, designated herein as SEQ ID NO:2892; reverse sequence CCTTGTGATTTTCCCCGTCT, designated herein as SEQ ID NO: 1064; probe sequence TGCTTTTGCCCCCTCGAATCTG, designated herein as SEQ ID NO: 2894), and RTS38622 (forward sequence AACCGGATCACCATTCCC, designated herein as SEQ ID NO: 2892; reverse sequence GTCTTCACCACGATGTTCCTC, designated herein as SEQ ID NO: 2896; probe sequence CACCAAGTCTGTGTCAGAAGGCCA, designated herein as SEQ ID NO: 2897) were used to measure RNA levels. GFAP RNA levels were normalized to total GAPDH, as measured by human primer probe set RTS104 (forward sequence GAAGGTGAAGGTCGGAGTC, designated herein as SEQ ID NO: 2898; reverse sequence GAAGATGGTGATGGGATTTC, designated herein as SEQ ID NO: 2899; probe sequence CAAGCTTCCCGTTCTCAGCC, designated herein as SEQ ID NO: 2900). Results are presented as percent of GFAP RNA, relative to untreated control cells (% UTC). The half maximal inhibitory concentration (IC₅₀) of each modified oligonucleotide was calculated using a linear regression on a log/linear plot of the data in Excel.

TABLE 113 Dose-dependent reduction of human GFAP RNA in U251 cells by modified oligonucleotides Compound GFAP RNA (% UTC) RTS38621 IC₅₀ No. 123 nM 370 nM 1100 nM 3300 nM 10000 nM (μM) 1072813 111 77 62 28 16 1.6 1166721 81 63 30 19 15 0.6 1166874 86 62 34 15 15 0.6 1166895 91 94 68 40 21 2.5 1166954 69 52 35 14 12 0.4 1047582 54 26 13 6 5 0.1

TABLE 114 Dose-dependent reduction of human GFAP RNA in U251 cells by modified oligonucleotides Compound GFAP RNA (% UTC) RTS38622 IC₅₀ No. 123 nM 370 nM 1100 nM 3300 nM 10000 nM (μM) 1072813 84 70 34 20 9 0.7 1166721 78 47 27 15 11 0.4 1166874 80 49 27 9 7 0.4 1166895 89 83 62 35 17 1.8 1166954 68 50 27 9 5 0.3 1047582 51 24 10 3 1 0.1

TABLE 115 Dose-dependent reduction of human GFAP RNA in U251 cells by modified oligonucleotides Compound GFAP RNA (% UTC) RTS38624 IC₅₀ No. 123 nM 370 nM 1100 nM 3300 nM 10000 nM (μM) 1072813 98 86 78 61 54 10.6 1166721 87 66 43 28 21 1.0 1166874 70 90 57 50 54 9.0 1166895 88 90 71 52 34 4.0 1166954 77 69 67 53 50 6.9 1047582 68 56 47 41 43 1.2

TABLE 116 Dose-dependent reduction of human GFAP RNA in U251 cells by modified oligonucleotides Compound GFAP RNA (% UTC) RTS38621 IC₅₀ No. 123 nM 370 nM 1100 nM 3300 nM 10000 nM (μM) 1166985 88 79 49 33 18 1.3 1166986 82 71 55 31 23 1.3 1166998 74 97 37 23 15 1.1 1167056 64 38 23 12 10 0.2 1199983 70 53 37 17 10 0.4 1048182 69 53 30 15 10 0.4

TABLE 117 Dose-dependent reduction of human GFAP RNA in U251 cells by modified oligonucleotides Compound GFAP RNA (% UTC) RTS38622 IC₅₀ No. 123 nM 370 nM 1100 nM 3300 nM 10000 nM (μM) 1166985 79 71 44 28 16 0.9 1166986 83 68 53 30 23 1.2 1166998 72 94 37 23 15 1.0 1167056 64 38 21 9 6 0.2 1199983 72 62 40 18 11 0.6 1048182 79 50 36 19 13 0.5

TABLE 118 Dose-dependent reduction of human GFAP RNA in U251 cells by modified oligonucleotides Compound GFAP RNA (% UTC) RTS38624 IC₅₀ No. 123 nM 370 nM 1100 nM 3300 nM 10000 nM (μM) 1166985 80 75 54 44 25 1.8 1166986 86 73 65 43 31 2.5 1166998 76 111 53 32 17 1.8 1167056 80 72 62 53 55 10.6 1199983 77 65 52 31 20 1.0 1048182 92 69 52 33 20 1.3

Example 11: Effect of Modified Oligonucleotides on Human GFAP RNA In Vitro, Multiple Doses

Modified oligonucleotides selected from the examples above were tested at various doses in U251 cells. Cultured U251 cells at a density of 20,000 cells per well were treated using free uptake with various concentrations of modified oligonucleotide as specified in the tables below. After a treatment period of approximately 48 hours, total RNA was isolated from the cells and GFAP RNA levels were measured by quantitative real-time RTPCR. Human GFAP primer probe sets RTS37485 (described herein above) was used to measure RNA levels. GFAP RNA levels were normalized to total GAPDH, as measured by human primer probe set RTS104 (described herein above). Results are presented as percent of GFAP RNA, relative to untreated control cells (% UTC). The half maximal inhibitory concentration (IC₅₀) of each modified oligonucleotide was calculated using a linear regression on a log/linear plot of the data in Excel.

TABLE 113 Dose-dependent reduction of human GFAP RNA in U251 cells by modified oligonucleotides Compound GFAP RNA (% UTC) RTS37485 IC₅₀ No. 250 nM 740 nM 2220 nM 6670 nM 20000 nM (μM) 1072813 105 102 80 59 34 10 1166721 92 83 64 49 32 7 1166874 81 78 55 38 22 4 1166895 83 89 85 78 73 950 1166926 97 93 79 73 58 31 1166927 89 86 78 76 52 28 1166954 92 90 77 67 58 29 1166985 104 108 104 94 80 43 1166986 94 92 95 84 82 1063 1166998 91 91 77 59 42 12 1167056 77 55 32 17 12 1 1167058 85 59 41 18 15 1 1199983 94 88 73 62 49 17 

1-43. (canceled)
 44. A modified oligonucleotide according to the following chemical structure:

or a salt thereof.
 45. A modified oligonucleotide according to the following chemical structure:

46-55. (canceled)
 56. The modified oligonucleotide of claim 44, which is the sodium salt or potassium salt of the chemical structure.
 57. A pharmaceutical composition comprising the modified oligonucleotide of claim 44 and a pharmaceutically acceptable carrier or diluent.
 58. The pharmaceutical composition of claim 57, wherein the pharmaceutically acceptable diluent is artificial cerebrospinal fluid.
 59. The pharmaceutical composition of claim 58, wherein the pharmaceutical composition consists of the compound and artificial cerebrospinal fluid.
 60. A compound comprising a modified oligonucleotide according to the following chemical notation: ^(m)C_(es)A_(eo)G_(eo)T_(eo)A_(eo)T_(eo)T_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds) ^(m)C_(ds)T_(ds)A_(ds) ^(m)C_(ds)T_(ds)A_(eo)G_(es)T_(es) ^(m)C_(es) (SEQ ID NO: 20), wherein: A=an adenine nucleobase, ^(m)C=a 5-methyl cytosine nucleobase, G=a guanine nucleobase, T=a thymine nucleobase, e=a 2′-β-D-MOE sugar moiety, d=a 2′-β-D-deoxyribosyl sugar moiety, s=a phosphorothioate internucleoside linkage, and o=a phosphodiester internucleoside linkage. 61-65. (canceled)
 66. The compound of claim 60, comprising the modified oligonucleotide covalently linked to a conjugate group.
 67. A pharmaceutical composition of claim 60, and a pharmaceutically acceptable diluent or carrier.
 68. The pharmaceutical composition of claim 67, wherein the pharmaceutically acceptable diluent is artificial cerebrospinal fluid.
 69. The pharmaceutical composition of claim 68, wherein the pharmaceutical composition consists of the compound and artificial cerebrospinal fluid. 70-78. (canceled)
 79. A method comprising administering to an individual the pharmaceutical composition of claim
 57. 80. A method of treating a disease associated with GFAP, comprising administering to an individual having or at risk of having a disease associated with GFAP a therapeutically effective amount of the pharmaceutical composition according to claim 57, thereby treating the disease associated with GFAP.
 81. (canceled)
 82. The method of claim 80, wherein the disease is a neurodegenerative disease.
 83. The method of claim 80, wherein the disease is Alexander disease.
 84. The method of claim 80, wherein at least one symptom or hallmark of the disease is ameliorated.
 85. The method of claim 84, wherein at least one symptom or hallmark is one or more of motor delays, cognitive delays, paroxysmal deterioration, seizures, vomiting, swallowing difficulties, ataxic gait, palatal myoclonus, autonomic dysfunction, or the presence of intra-astrocytic inclusions called Rosenthal fibers.
 86. The method of claim 80, wherein the pharmaceutical composition is administered to the central nervous system or systemically.
 87. The method of claim 80, wherein the pharmaceutical composition is administered to the central nervous system and systemically.
 88. (canceled)
 89. The pharmaceutical composition of claim 58, wherein the pharmaceutical composition consists essentially of the compound and artificial cerebrospinal fluid.
 90. The pharmaceutical composition of claim 68, wherein the pharmaceutical composition consists essentially of the compound and artificial cerebrospinal fluid. 